Cancer diagnosis and therapy

ABSTRACT

The present invention provides a novel approach to cancer diagnosis and therapy of cancer by providing compositions and methods for the identification and specific targeting of the cancer stem cell populations present in a tumour to eradicate, or slow or prevent tumour (5) growth and spread, including the potential for tumour metastasis, by modulation of the Renin-Angiotensin System including, but not limited to, Renin Receptor, Angiotensin II Receptor (2) and a secreted form of the Renin Receptor.

TECHNICAL FIELD

The present invention provides a novel approach to cancer diagnosis andcancer therapy. In particular, the identification and specific targetingof cancer stem cell populations present in a tumour to eradicate or slowor prevent tumour growth and spread, including the potential for tumourmetastasis, is contemplated within the scope of the present invention.The present invention is particularly useful in the identification andtreatment of tumours.

BACKGROUND OF THE INVENTION

Next to cardiovascular disease, cancer is one of the most significanthealth conditions worldwide that accounts for approximately one in fourdeaths. In the United States alone, health costs are estimated to runinto the hundreds of billions of dollars per annum, with around ahundred billion dollars in direct expenditures currently. Thisexpenditure is estimated to be up to US$207 billion by 2020. Theincidence of cancer is widely expected to increase as the populationages worldwide, further augmenting the impact of this spectrum ofdiseases. The current treatment regimens for cancer, established in the1970s and 1980s, have not changed dramatically. These treatments, whichinclude surgery, radiotherapy and chemotherapy, and other modalitiesincluding newer targeted therapies, have shown limited overall survivalbenefit when utilised in more advanced stage cancers since, among otherthings, these therapies primarily target the tumour bulk rather thancancer stem cells, which are thought to drive tumourigenesis.

Conventional cancer diagnosis and therapies to date have attempted toselectively detect and eradicate neoplastic cells that are largelyfast-growing (i.e., cells that form the tumour bulk). Standard cancertreatment regimens have often been largely designed to the deliver thehighest dose of radiation and/or administer chemotherapeutic agentwithout undue toxicity, i.e., often referred to as the “maximumtolerated dose” (MTD) or “no observed adverse effect level” (NOAEL).Chemotherapy is often added to radiotherapy to improve cancer control,at the expense of increased toxicities. Many conventional cancerchemotherapies (e.g., alkylating agents such as cyclophosphamide;antimetabolites such as 5-Fluorouracil; plant alkaloids such asvincristine) and conventional radiation therapies exert their toxiceffects on cancer cells largely by interfering with cellular mechanismsinvolved in cell growth and DNA replication. Chemotherapy protocols alsooften involve administration of a combination of chemotherapeutic agentsin an attempt to increase the efficacy of the treatment. Despite theavailability of a large variety of chemotherapeutic agents, thesetherapies have many limitations. For example, chemotherapeutic agentsare notoriously toxic due to non-specific effects on fast-growing cellswhether normal or malignant. For example, chemotherapeutic agents causesignificant, and often serious toxicities, including bone marrowdepression, immunosuppression, gastrointestinal distress, etc.

Other types of traditional cancer therapies include surgery, hormonaltherapy, immunotherapy, epigenetic therapy, anti-angiogenesis therapy,targeted therapy (e.g., therapy directed to a cancer target with agentssuch as Gleevec® and other tyrosine kinase inhibitors, Velcade®, Sutent®etc.), and radiation therapy to eradicate neoplastic cells in a patient.All of these approaches, often in combination, can pose significantdrawbacks for the patient including a lack of efficacy, toxicity andloss of quality of life. Accordingly, new and more effective therapiesand/or regimens for improving the long-term prospect including survivaland reduced side effects of treatment of cancer patients are needed.

Cancer stem cells comprise a unique subpopulation (typically ˜0.1-10%)of a tumour that, relative to the remaining 90% or so of the tumour(i.e., the tumour bulk), are more tumourigenic, relatively moreslow-growing or quiescent, and often more chemotherapy and/orradiotherapy resistant than the tumour cells. Given that conventionaltherapies and regimens have, in large part, been designed to attackrapidly proliferating cells (i.e., those cancer cells that comprise thetumour bulk), cancer stem cells which are often slow-growing arerelatively more resistant than faster growing tumour cells toconventional therapies and regimens. Furthermore, cancer stem cells maypossess other features that endow them with chemo-resistance such asmulti-drug resistance, and develop and/or enhance anti-apoptoticpathways. These features would constitute a key reason for the failureof standard cancer treatments to ensure long-term benefit in mostpatients especially those with more advanced-stage cancers (i.e., thefailure to adequately target and eradicate cancer stem cells). In someinstances, a cancer stem cell(s) is the founding cell of a tumour (i.e.,it is a progenitor giving rise to the cancer cells that comprise thetumour bulk).

Two models of cancer stem cell proliferation have been proposed. Thestochastic model postulates that oncogenic mutations occur randomly innormal cells and that every cell within a tumour has a low but equallikelihood of re-initiating a tumour. In contrast, the cancer stem cellmodel posits that tumours arise from a small, phenotypically distinctsubset of cancer cells that give rise to the heterogeneous cell lineagesobserved in a tumour.

Cancer stem cells have several properties that distinguish them from theremainder of the cancer cell population. Most importantly, they undergoasymmetrical cell division, a unique type of cell division in which oneoffspring cell remain identical to the parent cell, while the otherdifferentiates. In normal adult tissues, self-renewal is displayedexclusively by adult stem cells. Like embryonic stem cells, cancer stemcells sit on top of the tumour cell hierarchy and can respond to stimulito generate cells further along the differentiation spectrum, albeit inan aberrant manner. Cancer stem cells are also resistant to chemotherapyand radiotherapy, which could explain why conventional treatments areineffective in curing cancer and relapse occurs in the generally moreaggressive forms. Moreover, some cancer stem cells are relativelyquiescent shielding them from drugs that target highly proliferatingcells. Finally, cancer stem cells can result in metastasis in cancers.

Cancer stem cells have been identified in a large variety of cancertypes. For example, leukaemia cells bearing the specific phenotypeCD34⁺CD38⁻ (comprising <1% of a given leukaemia), unlike the remaining99+% of the leukaemia bulk, were able to recapitulate the leukaemia fromwhen it is derived when transferred into immunodeficient mice (Bonnet etal. (1997) Nat Med 3:730-737). That is, these cancer stem cells arefound as <1 in 10,000 leukaemia cells, yet this low frequency populationis able to initiate and serially transfer a human leukaemia with thesame histologic phenotype as in the original tumour into severe combinedimmunodeficiency/non-obese diabetic (NOD/SCID) mice.

Similar studies involving cancer stem cells isolated from, for example,human breast cancer (CD44⁺CD24^(low lin); Al-Hajj et al. (2003) ProcNat. Acad. Sci USA 100:3983-3988), human acute lymphoblastic leukaemia(CD34⁺CD10⁻, CC34⁺CD19⁻; Cox et al. (2004) Blood 104(19):2919-2925), andmultiple myeloma (CD138⁻; Matsui et al. (2004) Blood 103(6):2332) haveall been shown to have increased tumourigenic potential inrecapitulation studies in mice.

Since conventional cancer therapies target rapidly proliferating cells(i.e., cells that form the tumour bulk) these treatments are believed tobe relatively ineffective at targeting and impairing cancer stem cells.In fact, cancer stem cells, including leukaemia stem cells, have beenshown to be relatively resistant to conventional chemotherapeutic agents(e.g., Ara-C, Daunorubicin) as well as newer targeted therapies (e.g.,Gleevec®, Velcade®). For example, leukaemic stem cells are relativelyslow-growing or quiescent, express multi-drug resistance genes, andutilise other anti-apoptotic mechanisms, features which contribute totheir chemo-resistance. Further, by virtue of their chemo-resistance,cancer stem cells may contribute to treatment failure, and may alsopersist following treatment or recur at a later date following apparentinitial clinical remission.

Targeting cancer stem cells is expected to provide for improvedlong-term outcomes for cancer patients. Accordingly, a need exists toprovide new therapeutic agents and/or treatments designed to targetcancer stem cells to achieve more successful therapeutic outcomes. Thepresent invention seeks to address this problem.

SUMMARY OF THE INVENTION

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Summary of the Invention. It is not intended to beall-inclusive and the inventions described and claimed herein are notlimited to or by the features or embodiments identified in this Summaryof the Invention, which is included for purposes of illustration onlyand not restriction.

Applicants have identified discrete populations of cancer stem cellsthat have been shown to be associated with an extensive range ofdifferent tumour types, affecting the major organ systems examined.Accordingly, identification of these cancer stem cells and the cancerstem cell populations provides a novel approach to the management ofcancer, as well as in prognostic, diagnostic and follow-up applications.In addition, the Applicants have surprisingly demonstrated that thesecancer stem cells express markers associated with key regulatory systemsincluding, for example, the Renin-Angiotensin System (RAS) including thePro/Renin Receptor System (PRRS) and the associated bypass pathways.This novel insight provides a novel target and unique therapeuticopportunity in the management of cancer by employing established and/ornovel drugs that specifically target these regulatory pathways in anattempt to eradicate, or arrest growth, proliferation and/ordifferentiation of cancer stem cell populations. This has the potentialto reduce both the tumourigenic and metastatic potential of nascent andestablished tumours.

Accordingly, in one aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells in atumour within the cancer, wherein the cancer stem cells arecharacterised by (i) the expression of one or more embryonic stem cellbiomarkers, and (ii) the expression of one or more biomarkers associatedwith the Renin-Angiotensin System.

In another aspect of the present invention there is provided a methodfor preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells withinthe cancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more embryonic stem cell biomarkers, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System, and wherein the cancer is a solid cancer orblood cancer.

In yet another aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells withinthe cancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more embryonic stem cell biomarkers, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System, and wherein the tumour is selected from thegroup consisting of squamous cell carcinoma of the oral cavity, squamouscell carcinoma of the skin, melanoma, lung cancer, breast cancer, kidneycancer, brain cancer, bowel cancer, thyroid cancer, prostate cancer,lymphoma, leukaemia and sarcomas.

In yet a further aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells withinthe cancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more embryonic stem cell biomarkers, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System, and wherein the tumour is a squamous cellcarcinoma.

In another aspect of the present invention there is provided a methodfor preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells withinthe cancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more stem cell biomarker selected from the groupconsisting of Cripto, ABCG2, Alkaline Phosphatase/ALPL, CD9, FGF-4,GDF-3, Integrin alpha 6/CD49f, Integrin beta 1/CD29, NANOG, OCT-3/4,Podocalyxin, SOX2, SSEA-3, SSEA-4, STAT3, SSEA-1, FoxD3, DPPA5/ESG1,Rex-1/ZFP42, DPPA4, LIN-28A, UTF1, Lefty-A, Lefty-1, TBX3, ESGP,TRA-1-60(R), TRA-1-81, 5T4, TBX2, ZIC3, CD30/TNFRSF8, KLF5, c-Myc,GCNF/NR6A1, SUZ12, Smad2, CDX2, TROP-2, CD117/c-kit, LIN-41, Integrinalpha 6 beta 4, THAP11, Smad2/3, TBX5, TEX19, Oct-4A, TEX19.1, DPPA2,Activin RIB/ALK-4, Activin RIIB, FGF-5, GBX2, Stella/Dppa3, DNMT3B,F-box protein 15/FBXO15, LIN-28B, Integrin alpha 6 beta 1, KLF4, ERRbeta/NR3B2, EpCAM/TROP1, TERT, CHD1, Cbx2, c-Maf, L1TD1, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System.

In yet another aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells withinthe cancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more embryonic stem cell biomarker selected fromthe group consisting of OCT4, SOX2, NANOG and PSTAT3, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System selected from the group consisting of ReninReceptor (RR), Angiotensin II Receptor 2 and a secreted form of theRenin Receptor (sRR).

In yet a further aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent(s) tothe patient in an amount sufficient to selectively eradicate or, inhibitthe growth, proliferation and/or differentiation of cancer stem cellswithin the cancer, wherein the cancer stem cells are characterised by(i) the expression of one or more stem cell biomarker selected from thegroup consisting of Oct-4, SOX2, NANOG and PSTAT3, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System selected from the group consisting of ReninReceptor, Angiotensin II Receptor 2 and a secreted form of the ReninReceptor, and wherein the therapeutic agent is selected from the groupconsisting of Direct Renin Inhibitors (DRIs), Angiotensin-ConvertingEnzyme Inhibitors (ACEIs), Angiotensin Receptor Blockers (ARBs),Beta-Blockers, Cyclo-oxygenase 2 Inhibitors, Chymase Inhibitors,Inhibitors of Cathepsin B, Cathepsin D and Cathepsin G, Calcium, VitaminD, and Calcium Channel Blockers.

In yet another aspect of the present invention there is provided amethod for determining presence or absence of cancer in a subject, themethod comprising:

(i) detecting and/or measuring the levels of cancer stem cells presentin a biological sample obtained from the subject using biomarkerexpression analysis;

(ii) comparing the levels of the cancer stem cells obtained from thebiological sample against the cancer stem cell level from a controlpopulation;

wherein, an increased level in the cancer stem cells obtained from thebiological sample relative to the control population is diagnostic thatthe subject has, or is predisposed to developing, cancer.

In another aspect of the present invention there is provided a methodfor determining presence or absence of cancer in a subject, the methodcomprising:

(i) detecting and/or measuring the level of cancer stem cells in abiological sample obtained from the subject using biomarker expressionanalysis;

(ii) comparing the level of the cancer stem cells obtained from thebiological sample against the cancer stem cell level from a controlpopulation,

wherein, an increased level in the cancer stem cells obtained from thebiological sample relative to the control population is diagnostic thatthe subject has, or is predisposed to developing, cancer, and

(iii) administering a prophylactic or therapeutic regime(s) to thesubject who has, or is predisposed to developing, cancer.

In another aspect of the present invention there is provided apharmaceutical composition for use in a method for treatment of cancer,wherein the pharmaceutical composition comprises a therapeutic agentsufficient to selectively eradicate or, inhibit the growth,proliferation and/or differentiation of cancer stem cells within acancer, and wherein the method comprises administering the therapeuticagent to a patient with cancer.

In another aspect of the present invention there is provided a kit orarticle of manufacture for use in the treatment of cancer, the kitcomprising a therapeutic agent sufficient to selectively eradicate, orinhibit the growth, proliferation and/or differentiation of cancer stemcells within a cancer, together with instructions for how to administera therapeutic dose to the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the main pathways associated with the RAS. ACE: AngiotensinConverting Enzyme; ACEIs: Angiotensin Converting Enzyme inhibitors;Cox2i: Cox2 inhibitors; β-blockers: Beta-Blockers; ATIIR2: AngiotensinII Receptor 2; ATIIR1: Angiotensin II Receptor 1; (Pro)-RR: Pro(Renin)Receptors [also called Renin Receptor (RR)]; Vit D: Vitamin D; XX: majorblockades; ++: major promoting steps.

FIG. 2 shows the combined pathways associated with the RAS. ACE:Angiotensin Converting Enzyme; ACEI: Angiotensin Converting EnzymeInhibitors; Cox2i: Cox2 inhibitors; β-blockers: Beta-Blockers; ATIIR2:Angiotensin II Receptor 2; ATIIR1: Angiotensin II Receptor 1; (Pro)-RR:Pro(Renin) Receptors [also called Renin Receptor (RR)]; Vit D: VitaminD; XX: major blockades; x: minor blockades; ++: major promoting steps;+: minor blocking steps.

FIG. 3 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with oral tongue squamous cell carcinoma(OTSCC) as evidenced by individual immunohistochemical stainingprofiles.

FIGS. 4A and 4B show Western blot analysis of OTSCC cancer stem cellsusing antibodies specific for the RR, namely anti-ATP6IP2 primaryantibody (ab40790) and Goat anti-rabbit HRP secondary antibody (A16110).The predicted 39 kDa renin receptor protein band was present in bothOTSCC samples analysed. No staining was observed for cells associatedwith human liver tissue or secondary antibody alone (negative controls).

FIGS. 5A and 5B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with OTSCC. FIG. 5A showsimmunohistochemical co-staining using antibodies specific to OCT4 andRR. FIG. 5B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 6 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with melanoma as evidenced by theimmunohistochemical staining profiles.

FIGS. 7A and 7B shows co-localisation of OCT4 and RR by the cancer stemcell population associated with melanoma. FIG. 7A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 7B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 8 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with sarcoma (leiomyosarcoma) asevidenced by the immunohistochemical staining profiles.

FIGS. 9A and 9B shows the co-localisation of OCT4 and RR by cancer stemcell population associated with sarcoma. FIG. 9A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 9B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 10 shows expression of OCT4, SOX2, ATIIR2 and RR by the cancer stemcell population associated with bowel cancer as evidenced by theimmunohistochemical staining profiles.

FIGS. 11A and 11B shows the co-localisation of OCT4 and RR in the cancerstem cell population associated with bowel cancer. FIG. 11A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 11B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 12 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with brain cancer (glioblastomamultiforme) as evidenced by the immunohistochemical staining profiles.

FIGS. 13A and 13B shows the co-localisation of OCT4 and RR in the cancerstem cell population associated with brain cancer (glioblastomamultiforme). FIG. 13A shows immunohistochemical staining usingantibodies specific to OCT4 and RR. FIG. 13B shows quantification of therelative fluorescence signal for OCT4 (dots) and RR (long dashed line).

FIG. 14 shows the expression of OCT4, SOX2, ATIIR2 and RR in the cancerstem cell population associated with breast cancer as evidenced by theimmunohistochemical staining profiles.

FIGS. 15A and 15B shows the co-localisation of OCT4 and RR in the cancerstem cell population associated with breast cancer. FIG. 15A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 15B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 16 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with lung cancer (metastatic lungadenocarcinoma) as evidenced by the immunohistochemical stainingprofiles.

FIGS. 17A and 17B shows the co-localisation of OCT4 and RR in cancerstem cell population associated with lung cancer (metastatic lungadenocarcinoma). FIG. 17A shows immunohistochemical staining usingantibodies specific to OCT4 and RR. FIG. 17B shows quantification of therelative fluorescence signal for OCT4 (dots) and RR (long dashed line).

FIG. 18 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with B cell lymphoma as evidenced by theimmunohistochemical staining profiles.

FIGS. 19A and 19B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with B cell lymphoma. FIG. 19A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 19B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 20 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with kidney cancer (metastatic renalcell cancer) as evidenced by the immunohistochemical staining profiles.

FIGS. 21A and 21B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with kidney cancer (metastatic renalcell cancer). FIG. 21A shows immunohistochemical staining usingantibodies specific to OCT4 and RR. FIG. 21B shows quantification of therelative fluorescence signal for OCT4 (dots) and RR (long dashed line).

FIG. 22 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with thyroid cancer as evidenced by theimmunohistochemical staining profiles.

FIGS. 23A and 23B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with thyroid cancer. FIG. 23A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 23B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 24 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with chronic lymphocytic leukaemia asevidenced by the immunohistochemical staining profiles.

FIGS. 25A and 25B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with chronic lymphocytic leukaemia. FIG.25A shows immunohistochemical staining using antibodies specific to OCT4and RR. FIG. 25B shows quantification of the relative fluorescencesignal for OCT4 (dots) and RR (long dashed line).

FIG. 26 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with skin squamous cell carcinoma asevidenced by the immunohistochemical staining profiles.

FIGS. 27A and 27B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with skin squamous cell carcinoma. FIG.27A shows immunohistochemical staining using antibodies specific to OCT4and RR. FIG. 27B shows quantification of the relative fluorescencesignal for OCT4 (dots) and RR (long dashed line).

FIG. 28 shows the expression of OCT4, SOX2, ATIIR2 and RR by the cancerstem cell population associated with prostate cancer as evidenced by theimmunohistochemical staining profiles.

FIGS. 29A and 29B shows the co-localisation of OCT4 and RR by the cancerstem cell population associated with prostate cancer. FIG. 29A showsimmunohistochemical staining using antibodies specific to OCT4 and RR.FIG. 29B shows quantification of the relative fluorescence signal forOCT4 (dots) and RR (long dashed line).

FIG. 30 shows the expression of OCT4 and SOX2 in a human seminoma tissuesample, ATIIR2 in human kidney and RR in human placental tissues asrespective positive controls. The negative control shows absence ofstaining without the primary antibody in a brain cancer (glioblastomamultiforme) tissue section.

SELECTED DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions belong. Although any assays, methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, various assays,methods, devices and materials are now described.

It is intended that reference to a range of numbers disclosed herein(for example 1 to 10) also incorporates reference to all related numberswithin that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9and 10) and also any range of rational numbers within that range (forexample 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, allsub-ranges of all ranges expressly disclosed herein are expresslydisclosed. These are only examples of what is specifically intended andall possible combinations of numerical values between the lowest valueand the highest value enumerated are to be considered to be expresslystated in this application in a similar manner.

As used in this specification, the words “comprises”, “comprising”, andsimilar words, are not to be interpreted in an exclusive or exhaustivesense. In other words, they are intended to mean “including, but notlimited to”.

As used herein, the term “antibodies” refer to molecules that contain anantigen binding site, e.g., immunoglobulins. Immunoglobulin moleculescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Antibodies include,but are not limited to, monoclonal antibodies, polyclonal antibodies,multispecific antibodies, human antibodies, humanised antibodies, murineantibodies, camelised antibodies, chimeric antibodies, single domainantibodies, single chain Fvs (scFv), single chain antibodies, Fabfragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), andanti-idiotopic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above.

As used herein, the term “cancer” refers to a neoplasm or tumourresulting from abnormal uncontrolled growth of cells. The term “cancer”encompasses a disease involving both pre-malignant and malignant cancercells. In some examples, cancer refers to a localised overgrowth ofcells that has not spread to other parts of a subject, i.e., a benigntumour. In other examples, cancer refers to a malignant tumour, whichhas invaded and destroyed neighboring body structures and/or spread todistant sites.

As used herein, the term “cancer cells” refer to cells that acquire acharacteristic set of functional capabilities during their development,including the ability to evade apoptosis, are self-sufficienct in growthsignals and are insensitivite to anti-growth signals, tissueinvasion/metastasis, significant growth potential, and/or sustainedangiogenesis. The term “cancer cell” is meant to encompass bothpre-malignant and malignant cancer cells.

As used herein, the term “cancer stem cell(s)” refers to a cell that canbe a progenitor of a highly proliferative cancer cell. A cancer stemcell has the ability for assymmetrical division and to re-grow a tumouras demonstrated by its ability to form tumours in immunocompromisedmice, and typically to form tumours upon subsequent serialtransplantation in immunocompromised mice. Cancer stem cells are alsotypically slow-growing relative to the bulk of a tumour; that is, cancerstem cells are generally quiescent. In certain examples, but not all,the cancer stem cell may represent approximately 0.1 to 10% of a tumour.

As used herein, the term “cancer stem cell population” is intended tomean one or more cancer stem cells, in other words a single cancer stemcell or multiple cancer stem cells, the single cancer stem cell ormultiple cancer stem cells being capable of driving tumourigenesis of agiven cancer.

As used herein, the term “squamous cell carcinomas” refers to theepithelial tumours found in many different organs, including the skin,upper aerodigestive tract (including oral cavity) and paranasal sinuses,oesophagus, lungs, and cervix, and other organs which show squamous celldifferentiation. Included are head and neck squamous cell carcinomas,lung squamous cell carcinomas, skin squamous cell carcinomas, oticsquamous cell carcinomas, vulval squamous cell carcinomas, cervicalsquamous cell carcinomas, oesophageal squamous cell carcinomas, upperaerogigestive tract and paranasal sinus squamous cell carcinomas and thelike.

As used herein, the term “Renin-Angiotensin System (RAS)” or“Renin-Angiotensin-Aldosterone System (RAAS)” is a hormone system thatregulates blood pressure and fluid balance. The wider pathway associatedwith RAS also includes the Pro/Renin Receptor System (PRRS) and theassociated bypass pathways. By way of example, refer to FIGS. 1 and 2.There are a number of known drugs which target the RAS including PRRS,as described in more detail below.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of cancer and one or more symptomsthereof, to enhance or improve the prophylactic effect(s) of anothertherapy, reduce the severity, the duration of cancer, ameliorate one ormore symptoms of cancer, prevent the advancement of cancer, causeregression of cancer, and/or enhance or improve the therapeuticeffect(s) of another therapy. In an example of the invention, the amountof a therapy is effective to achieve one, two or three or more resultsfollowing the administration of one, two, three or more therapies: (1) astabilisation, reduction or eradication of the cancer stem cellpopulation; (2) a stabilisation, reduction or eradication in the cancercell population; (3) a stabilisation or reduction in the growth of atumour or neoplasm; (4) an impairment in the formation of a tumour; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalisation rate, (10) a decrease in hospitalisation lengths, (11)the size of the tumour is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%, (12) an increase in the number of patients inremission, (13) an increase in the length or duration of remission, (14)a decrease in the recurrence rate of cancer, (15) an increase in thetime to recurrence of cancer, and (16) an amelioration of cancer-relatedsymptoms and/or quality of life.

As used herein, the terms “manage”, “managing”, and “management” in thecontext of the administration of a therapy to a subject refer to thebeneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic agent) or a combination of therapies, whilenot resulting in a cure of cancer. In certain examples, a subject isadministered one or more therapies (e.g., one or more prophylactic ortherapeutic agents) to “manage” cancer so as to prevent the progressionor worsening of the condition.

As used herein, the terms “prevent”, “preventing” and “prevention” inthe context of the administration of a therapy to a subject refers tothe prevention or inhibition of the recurrence, onset, and/ordevelopment of a cancer or a symptom thereof in a subject resulting fromthe administration of a therapy (e.g., a prophylactic or therapeuticagent), or a combination of therapies (e.g., a combination ofprophylactic or therapeutic agents). In some examples, such terms referto one, two, three or more results following the administration of oneor more therapies: (1) a stabilisation, reduction or eradication of thecancer stem cell population, (2) a stabilisation, reduction oreradication of the cancer cell population, (3) an increase in theresponse rate, (4) an increase in the duration of remission, (5) adecrease in the recurrence rate of cancer, (6) an increase in the timeto recurrence of cancer, (7) an increase in the disease-free,relapse-free, progression-free, and/or overall survival of the patient,and (8) an amelioration of cancer-related symptoms and/or quality oflife. In specific examples, such terms refer to a stabilisation,reduction or eradication of the cancer stem cell population.

As used herein, the term “marker” or “biomarker” in the context of atissue (e.g. a normal cell or tumour cell) means any antigen, moleculeor other chemical or biological entity that is specifically found in oron a tissue that it is desired to be identified or identified in or on aparticular tissue affected by a disease or disorder, for example cancer.The term “tumourigenic biomarker” is also relevant to this definition inthe context of cancer. In specific examples, the marker is a cellsurface antigen that is differentially or preferentially expressed byspecific cell types. In specific examples, the marker is a nuclearantigen that is differentially or preferrentially expressed by specificcell types. In specific examples the marker is an intracellular antigenthat is differentially or preferrentially expressed by specific celltypes.

As used herein, the term “prophylactic agent” refers to any molecule,compound, and/or substance that is used for the purpose of preventingcancer. Examples of prophylactic agents include, but are not limited to,proteins, immunoglobulins (e.g., multi-specific Igs, single chain Igs,Ig fragments, polyclonal antibodies and their fragments, monoclonalantibodies and their fragments), antibody conjugates or antibodyfragment conjugates, peptides (e.g., peptide receptors, selectins),binding proteins, chemospecific agents, chemotoxic agents (e.g.,anti-cancer agents), proliferation based therapy, and small moleculedrugs.

As used herein, the term “therapeutic agent” refers to any molecule,compound, and/or substance that is used for the purpose of treatingand/or managing a disease or disorder. Examples of therapeutic agentsinclude, but are not limited to, proteins, immunoglobulins (e.g.,multi-specific Igs, single chain Igs, Ig fragments, polyclonalantibodies and their fragments, monoclonal antibodies and theirfragments), peptides (e.g., peptide receptors, selectins), bindingproteins, biologies, chemospecific agents, chemotoxic agents (e.g.,anti-cancer agents), proliferation-based therapy agents, hormonalagents, radioimmunotherapies, targeted agents, epigenetic therapies,differentiation therapies, biological agents, radiation agents,chemotherapy, anti-angiogenic agents, and small molecule drugs.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a cancer or one or moresymptoms thereof. In certain examples, the terms “therapy” and“therapies” refer to chemotherapy, radiation therapy, surgery, hormonaltherapy, anti-angiogenic therapy, biological therapy, proliferationbased therapy, prodrug-activating enzyme therapy, small moleculetherapy, toxin therapy, antibody therapy, immunotherapy,radioimmunotherapy, targeted therapy, epigenetic therapy, demethylationtherapy, histone deactylase inhibitor therapy, differentiation therapyand/or other therapies useful in the prevention, management and/ortreatment of a cancer or one or more symptoms thereof.

As used herein, the terms “treat”, “treatment” and “treating” in thecontext of the administration of a therapy to a subject refer to thereduction or inhibition of the progression and/or duration of cancer,the reduction or amelioration of the severity of cancer, and/or theamelioration of one or more symptoms thereof resulting from theadministration of one or more therapies. In specific examples, suchterms refer to one, two or three or more results following theadministration of one, two, three or more therapies: (1) astabilization, reduction or eradication of the cancer stem cellpopulation; (2) a stabilisation, reduction or elimination in the cancercell population; (3) a stabilisation or reduction in the growth of atumour or neoplasm; (4) an impairment in the formation of a tumour; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalisation rate, (10) a decrease in hospitalisation lengths, (11)the size of the tumour is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%, and (12) an increase in the number of patientsin remission. In certain examples, such terms refer to a stabilisationor reduction in the cancer stem cell population. In some examples, suchterms refer to a stabilisation or reduction in the growth of cancercells. In some examples, such terms refer to a stabilisation orreduction in the cancer stem cell population and a reduction in thecancer cell population. In some examples, such terms refer to astabilisation or reduction in the growth and/or formation of a tumour.In some examples, such terms refer to the eradication, removal, orcontrol of primary, regional, or metastatic cancer (e.g., theminimisation or delay of the spread of cancer). In some examples, suchterms refer to a reduction in mortality and/or an increase in survivalrate of a patient population. In further examples, such terms refer toan increase in the response rate, the durability of response, or numberof patients who respond or are in remission. In some examples, suchterms refer to a decrease in hospitalisation rate of a patientpopulation and/or a decrease in hospitalisation length for a patientpopulation.

The term “sample” or “biological sample” as used herein means any sampletaken or derived from a subject. Such a sample may be obtained from asubject, or may be obtained from biological materials intended to beprovided to the subject. For example, a sample may be obtained fromblood being assessed, for example, to investigate the cancer status of asubject. Included are samples taken or derived from any subjects such asfrom normal healthy subjects and/or healthy subjects for whom it isuseful to understand their cancer status. Preferred samples arebiological fluid samples. The term “biological fluid sample” as usedherein refers to a sample of bodily fluid obtained for the purpose of,for example, diagnosis, prognosis, classification or evaluation of asubject of interest, such as a patient. In certain embodiments, such asample may be obtained for the purpose of determining the cancer statusof a patient. The sample may be any sample known in the art in whichcancer stem cells may be detected. Included are any body fluids such asa whole blood sample, plasma, serum, ovarian follicular fluid sample,seminal fluid sample, cerebrospinal fluid, saliva, sputum, urine,pleural effusions, interstitial fluid, synovial fluid, lymph, tears, forexample, although whole blood sample, plasma and serum are particularlysuited for use in this invention. In addition, one of skill in the artwould realise that certain body fluid samples would be more readilyanalysed following a fractionation or purification procedure, forexample, separation of whole blood into serum or plasma components.

The term “purified” as used herein does not require absolute purity.Purified refers in one embodiment to at least 90%, or 95%, or 98%, or99% homogeneity of, to provide an example, of a polypeptide or antibodyin a sample.

The term “subject” as used herein is preferably a mammal and includeshuman, and non-human mammals such as cats, dogs, horses, cows, sheep,deer, mice, rats, primates (including gorillas, rhesus monkeys andchimpanzees), possums and other domestic farm or zoo animals. Thus, theassays, methods and kits described herein have application to both humanand non-human animals, in particular, and without limitation, humans,primates, farm animals including cattle, sheep, goats, pigs, deer,alpacas, llamas, buffalo, companion and/or pure bred animals includingcats, dogs and horses. Preferred subjects are humans, and mostpreferably “patients” who as used herein refer to living humans who mayreceive or are receiving medical care or assessment for a disease orcondition. Further, while a subject is preferably a living organism, theinvention described herein may be used in post-mortem analysis as well.

The term “ELISA” as used herein means an enzyme linked immunosorbentassay, a type of competitive binding assay comprising antibodies and adetectable label used to quantitate the amount of an analyte in asample.

The term “capture antibody” as used herein means an antibody which istypically immobilized on a solid support such as a plate, bead or tube,and which antibody binds to and captures analyte(s) of interest, forexample membrane bound markers associated with a cancer stem cellpopulation.

The term “detection antibody” as used herein means an antibodycomprising a detectable label that binds to analyte(s) of interest. Thelabel may be detected using routine detection means for a quantitative,semi-quantitative or qualitative measure of the analyte(s) of interest,for example membrane bound markers associated with a cancer stem cellpopulation.

As used herein, the term “relating to the presence or amount” of ananalyte reflects that assay signals are typically related to thepresence or amount of an analyte through the use of a standard curvecalculated using known concentrations of the analyte of interest. As theterm is used herein, an assay is “configured to detect” an analyte if anassay can generate a detectable signal indicative of the presence oramount of a physiologically relevant concentration of the analyte.Typically, an analyte is measured in a sample.

A level “higher” or “lower” than a control, or a “change” or “deviation”from a control (level) in one embodiment is statistically significant. Ahigher level, lower level, deviation from, or change from a controllevel or mean or historical control level can be considered to exist ifthe level differs from the control level by about 5% or more, by about10% or more, by about 20% or more, or by about 50% or more compared tothe control level. Statistically significant may alternatively becalculated as P≤0.05. Higher levels, lower levels, deviation, andchanges can also be determined by recourse to assay reference limits orreference intervals. These can be calculated from intuitive assessmentor non-parametric methods. Overall, these methods may calculate the0.025, and 0.975 fractiles as 0.025*(n+1) and 0.975 (n+1). Such methodsare well known in the art. Presence of a marker absent in a control maybe seen as a higher level, deviation or change. Absence of a markerpresent in a control may be seen as a lower level, deviation or change.

DETAILED DESCRIPTION

There is an emerging concept that cancer stem cells drive thepersistence or recurrence of a tumour. Conventional cancer therapies,while successful in eradicating the bulk of tumours, are typically lesseffective on insidious cancer stem cells. Further, selective drugresistance exhibited by these cells contributes to significant morbidityand mortality in cancer sufferers. Accordingly, there is a need fortherapeutic regimes that specifically and selectively target cancer stemcells.

The present invention is predicated on the surprising and unexpecteddiscovery that discrete cancer stem cell populations are associated withcertain tumours including (e.g., squamous cell carcinoma of the oralcavity, squamous cell carcinoma of the skin, melanoma, lung cancer,breast cancer, kidney cancer, brain cancer, bowel cancer, thyroidcancer, prostate cancer, lymphoma, leukaemia and sarcomas. The cancerstem cell populations associated with these tumours are characterised byunique biomarker expression profiles that allows for the specificidentification and diagnosis of certain cancers.

Importantly, it has also been revealed by the Applicants that thesecancer stem cell populations express key components of theRenin-Agiotensin System (RAS), including the Renin Receptor (RR)Angiotensin II Receptor 2 (ATIIR2), as well as a secreted form of theRenin Receptor (sRR). In reference to FIGS. 3, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26 and 28, the Applicants demonstrate co-expression of RRand ATIIR2 by the cancer stem cell populations associated with varioustumours. These cancer stem cell populations are characterised by, forexample, the expression of OCT4, SOX2, PSTAT3 and NANOG. Accordingly,the expression of the components of RAS by these cancer stem cellpopulations provides a novel and unique therapeutic approach bytargeting the cancer stem cells associated with various tumours from theextensive array of drugs that target RAS such as, Angiotensin-ConvertingEnzyme Inhibitors (ACEis), Angiotensin Receptor Blockers (ARBs), DirectRenin Inhibitors (DRIs), Beta-Blockers, Cyclo-oxygenase 2 Inhibitors,Chymase Inhibitors, Inhibitors of Cathepsin B, Cathepsin D and CathepsinG, Calcium Supplements, Vitamin D and Calcium Channel Blockers.

In addition, the present invention also contemplates indirect inhibitorsof the RAS (e.g., Calcium Channel Blockers).

Accordingly, in one aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eliminate or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells in atumour associated with the cancer, wherein the cancer stem cells arecharacterised by (i) the expression of one or more embryonic stem cellbiomarkers, and (ii) the expression of one or more biomarkers associatedwith the Renin-Angiotensin System.

In another aspect of the present invention there is provided a methodfor preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively inhibit the growth,proliferation and/or differentiation of cancer stem cells within thecancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more embryonic stem cell biomarkers, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System, and wherein the cancer is a solid tumour orblood cancer.

In one example, the one or more embryonic stem cell markers is selectedfrom the group consisting of Cripto, ABCG2, Alkaline Phosphatase/ALPL,CD9, FGF-4, GDF-3, Integrin alpha 6/CD49f, Integrin beta 1/CD29, Nanog,Oct-3/4, Podocalyxin, SOX2, SSEA-3, SSEA-4, STAT3, SSEA-1, FoxD3,DPPA5/ESG1, Rex-1/ZFP42, DPPA4, LIN-28A, UTF1, Lefty-A, Lefty-1, TBX3,ESGP, TRA-1-60(R), TRA-1-81, 5T4, TBX2, ZIC3, CD30/TNFRSF8, KLF5, c-Myc,GCNF/NR6A1, SUZ12, Smad2, CDX2, TROP-2, CD117/c-kit, LIN-41, Integrinalpha 6 beta 4, THAP11, Smad2/3, TBX5, TEX19, Oct-4A, TEX19.1, DPPA2,Activin RIB/ALK-4, Activin RIIB, FGF-5, GBX2, Stella/Dppa3, DNMT3B,F-box protein 15/FBXO15, LIN-28B, Integrin alpha 6 beta 1, KLF4, ERRbeta/NR3B2, EpCAM/TROP1, TERT, CHD1, Cbx2, c-Maf and L1TD1. In anotherexample, the one or more embryonic stem cell biomarkers consists inOCT4, SOX2, NANOG and PSTAT3. In yet another example, the one or morebiomarkers associated with the RAS is selected from the group consistingof RR, ATIIR2, and sRR.

In yet another aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells withinthe cancer, wherein the cancer stem cells are characterised by (i) theexpression of one or more embryonic stem cell biomarkers, and (ii) theexpression of one or more biomarkers associated with theRenin-Angiotensin System, and wherein the tumour is selected from thegroup consisting of squamous cell carcinoma of the oral cavity, squamouscell carcinoma of the skin, melanoma, lung cancer, breast cancer, kidneycancer, brain cancer, bowel cancer, thyroid cancer, prostate cancer,lymphoma, leukaemia and sarcomas.

In yet a further aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells in atumour associated with the cancer, wherein the cancer stem cells arecharacterised by (i) the expression of one or more embryonic stem cellbiomarkers, and (ii) the expression of one or more biomarkers associatedwith the Renin-Angiotensin System, and wherein the tumour is a squamouscell carcinoma.

In another aspect of the present invention there is provided a methodfor preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eliminate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells in atumour associated with the cancer, wherein the cancer stem cells arecharacterised by (i) the expression of one or more stem cell biomarkerselected from the group consisting of Cripto, ABCG2, AlkalinePhosphatase/ALPL, CD9, FGF-4, GDF-3, Integrin alpha 6/CD49f, Integrinbeta 1/CD29, NANOG, OCT3/4, Podocalyxin, SOX2, SSEA-3, SSEA-4, STAT3,SSEA-1, FoxD3, DPPA5/ESG1, Rex-1/ZFP42, DPPA4, LIN-28A, UTF1, Lefty-A,Lefty-1, TBX3, ESGP, TRA-1-60(R), TRA-1-81, 5T4, TBX2, ZIC3,CD30/TNFRSF8, KLF5, c-Myc, GCNF/NR6A1, SUZ12, Smad2, CDX2, TROP-2,CD117/c-kit, LIN-41, Integrin alpha 6 beta 4, THAP11, Smad2/3, TBX5,TEX19, Oct-4A, TEX19.1, DPPA2, Activin RIB/ALK-4, Activin RIIB, FGF-5,GBX2, Stella/Dppa3, DNMT3B, F-box protein 15/FBXO15, LIN-28B, Integrinalpha 6 beta 1, KLF4, ERR beta/NR3B2, EpCAM/TROP1, TERT, CHD1, Cbx2,c-Maf and L1TD1, and (ii) the expression of one or more biomarkersassociated with the Renin-Angiotensin System.

In yet another aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eliminate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells in atumour associated with the cancer, wherein the cancer stem cells arecharacterised by (i) the expression of one or more stem cell markerselected from the group consisting of OCT4, SOX2, NANOG and PSTAT3, and(ii) the expression of one or more biomarkers associated with theRenin-Angiotensin System selected from the group consisting of ReninReceptor, Angiotensin II Receptor 2, and a secreted for of the ReninReceptor.

In yet a further aspect of the present invention there is provided amethod for preventing, treating, or managing cancer in a patient in needthereof, the method comprising administering a therapeutic agent to thepatient in an amount sufficient to selectively eradicate, or inhibit thegrowth, proliferation and/or differentiation of cancer stem cells in atumour associated with the cancer, wherein the cancer stem cells arecharacterised by (i) the expression of one or more stem cell biomarkerselected from the group consisting of OCT4, SOX2, NANOG and PSTAT3, and(ii) the expression of Renin Receptor, Angiotensin II Receptor 2 and/ora secreted form of Renin Receptor, and wherein the therapeutic agent isselected from the group consisting of Direct Renin Inhibitors (DRIs),ACEis, ARBs, Beta-Blockers, Cyclo-oxygenase 2 Inhibitors, ChymaseInhibitors, Inhibitors of Cathepsin B, Cathepsin D and Cathepsin G,Calcium, Vitamin D, and Calcium Channel Blockers.

In one example, the cancer stem cells are characterised by theexpression of SOX2, OCT4, PSTAT3 and NANOG. These cells are said to havea marker expression profile: SOX2⁺Oct-4⁺PSTAT3⁺NANOG⁺.

In a related example, the cancer stem cells are cancer stem cells ofsquamous cell carcinoma of oral tongue and are characterised by themarker expression profile CD44⁺SOX2⁺OCT4⁺NANOG⁺. In a further example,the cancer stem cells of squamous cell carcinoma of oral tongue arecharacterised by the marker expression profileCD44⁺SOX2⁺OCT4⁺NANOG⁺CD34⁻. In yet a further example, the cancer stemcells are cancer stem cells of squamous cell carcinoma of oral tongueand are characterised by the marker expression profileCD44⁺SOX2⁺OCT4⁺NANOG⁺CD34⁻p63⁻EMA⁻.

The cancer stem cells may co-express with embryonic stem cell markers,lymphatic cell markers, epithelial cancer cell markers or anycombination thereof. Accordingly, in one example, the cancer stem cellsco-express with embryonic stem cell markers and epithelial cancer cellmarkers. In another example, the cancer stem cells co-express withlymphatic cell markers and epithelial cancer cell markers. In a furtherexample, the cancer stem cells co-express with embryonic stem cellmarkers, lymphatic cell markers, and epithelial cancer cell markers.

While the present invention is particularly relevant to solid tumours,it also extends to blood cancers. For example, the Applicantsdemonstrate that Chronic Lymphocytic Leukaemia comprise cancer stemcells as characterised by the expression of OCT4, SOX2, ATIIR2 and RR(FIG. 24), which cancer stem cells also co-express the RR and ATIIR2(FIGS. 25A and 25B). Accordingly, the therapeutic methods andcompositions according to the present invention extend to blood cancersas well.

Accordingly, the present invention provides compositions and methodsrelated to identifying and targeting the growth and proliferation ofcancer stem cells as the cause of tumour growth, spread and metastasis.In particular, the compositions and methods are directed to cancer stemcells which display unique tumouriogenic biomarker expression profiles.By specifically targeting cancer stem cells, it is assumed that thetumourigenic and metastatic potential of the (nascent or established)tumour is significantly diminished, thereby leading to enhancedtherapeutic outcomes.

The cancer stem cells may be associated with a variety of cancers,including but not limited to, squamous cell carcinoma of the upperaerodigestive tract (including oral cavity), squamous cell carcinoma ofthe skin, melanoma, lung cancer, breast cancer, kidney cancer, braincancer, bowel cancer, thyroid cancer, prostate cancer, lymphoma,leukemia and sarcomas.

Squamous cell carcinomas include head and neck squamous cell carcinomas(including squamous cell carcinomas of the upper aerodigestive tract[including oral cavity] and paranasal sinuses and elsewhere),oesophageal squamous cell carcinomas, skin squamous cell carcinomas,lung squamous cell carcinomas, vulval squamous cell carcinomas andcervical squamous cell carcinomas. In one example the upperaerodigestive tract squamous cell carcinoma is squamous cell carcinomaof oral tongue.

The present invention provides methods for preventing, treating, and/ormanaging cancer, the method comprising administering to a subject inneed thereof a course of therapy that stabilises, reduces, or eradicatethe cancer stem cell population. In certain examples, the stabilisation,reduction, or elimination of the cancer stem cell population is achievedby administering a therapy that targets the growth and proliferation ofthe cancer stem cells.

Surprisingly, Applicants demonstrate that the cancer stem cellpopulations identified in the methods according to the present inventionco-express components of RAS, and in cancer stem cell populationsassociated with multiple different tumour types. By way of illustrationonly, the Applicants demonstrate co-expression of the RR and ATIIR2 inOTSCC (FIGS. 4A, 4B), melanoma (FIGS. 7A, 7B), sarcoma (FIGS. 9A, 9B),bowel cancer (FIGS. 11A, 11B), brain cancer (FIGS. 13A, 13B), breastcancer (FIGS. 15A, 15B), lung cancer (FIGS. 17A, 17B), B cell lymphoma(FIGS. 19A, 19B), and kidney (FIGS. 21A, 21B), thyroid cancer (23A,23B), chronic lymphocytic cancer (25A, 25B), skin squamous cellcarcinoma (27A, 27B), prostate cancer (29A, 29B).

Accordingly, therapy that targets the growth and proliferation of cancerstem cell populations comprises administering a therapeutic agent thatselectively targets components of the RAS and/or Pro/Renin ReceptorSystems (PRRS) expressed by the cancer stem cells. FIGS. 1 and 2 showthe types of inhibitors/drugs that target these systems, useful inaccordance with the compositions and methods according to the presentinvention.

Examples of known therapeutics that target the Renin-Angiotensin Systeminclude, but are not limited to, ACEIs, ARBs, DRIs, Beta-Blockers,Cyclo-oxygenase 2 Inhibitors, Inhibitors of Cathepsin B, Cathepsin D andCathepsin G, Calcium Channel Blockers, Calcium Supplements and VitaminD.

Examples of ACEIs include, but are not limited to, Benazepril (Lotesin),Captopril (Capoten), Cilazipril, Enalapril (Vasotec, Renitec),Fosinopril (Monopril), Lisinopril (Lisodur, Lopril, Novatec, Prinivil,Zestril), Moexipril, Perindopril (Coversay, Aceon), Quinapril(Accupril), Ramipril (Altace, Tritace, Ramace, Ramiwin), Trandolapril,Delapril, Zofenopril and Imidapril.

Examples of ARBs include, but are not limited to, Losartan, Irbesartan,Candesartan, Eprosartan, Olmesartan, Telmisartan, PD123319 andValsartan.

Examples of Beta-Blockers include, but are not limited to, Acebutolol(Sectral), Atenolol (Tenormin), Betaxolol (Betoptic), Bisoprolol(Cardicor, Emcor, Zebeta), Carteolol (Teoptic), Carvedilol (Coreg,Eucardic), Celiprolol (Celectol), Labetalol (Trandate), Levobunolol(Betagan), Metipranolol (Metipranolol Minims), Metoprolol (Betaloc,Lopresor, Lopressor, Toprol XL), Nadolol (Corgard), Nebivolol (Bystolic,Nebilet), Oxprenolol (Trasicor), Pindolol (Visken), Propranolol (InderalLA), Sotalol (Beta-Cardone, Sotacor), and Timolol (Betim, Nyogel,Timoptol).

Examples of Cyclo-oxygenase 2 Inhibitors include, but are not limitedto, Celecoxib, Nepafenac, Ibuprofen (Dolgesic), Indomethacin, Sulindac,Xanthohumol, Meclofenamate Sodium, Meloxicam, Rofecoxib, BromfenacSodium, Ibuprofen Lysine, Ketorolac (Ketorolac tromethamine), DiclofenacSodium, Etodolac, Ketoprofen, Naproxen Sodium, Piroxicam, Acemetacin,Phenacetin, Tolfenamic Acid, Nimesulide, Flunixin Meglumin, Aspirin,Bufexamac, Niflumic acid, Licofelone, Oxaprozin, Lornoxicam,Lumiracoxib, Zaltoprofen, Ampiroxicam, Valdecoxib, Nabumetone, MefenamicAcid, Carprofen, Amfenac Sodium monohydrate, Asaraldehyde and Suprofen.

Examples of Chymase Inhibitors include, but are not limited to, TY-51469(2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylicacid), Eglin C, CI, SUN13834, Chymostatin, TJK002 a benzimidazoleinhibitor, ONO-WH-236, Amblyomma americanum tick serine proteaseinhibitor 6 (AamS6), N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK),Alpha-aminoalkylphosphonate diaryl esters, Serine protease inhibitor A3(serpinA3), Squamous cell carcinoma antigen (SCCA-2), Bortezomib(Velcade), RO5066852 and 17beta-estradiol.

Examples of Cathepsin B Inhibitors include, but are not limited to,Cystatin B, Cystatin C, Cysteine peptidase inhibitor E64,[Pt(dmba)(aza-N1)(dmso)] complex 1 (a potential anti-tumoral drug withlower IC50 than cisplatin in several tumoral cell lines),2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), CA-074Me, Lipidated CtsBinhibitor incorporated into the envelope of a liposomal nanocarrier(LNC-NS-629), Proanthocyanidin (PA) and ahpatinin Ac (1) and ahpatininPr (2).

Examples of Cathepsin D Inhibitors include, but are not limited to,non-peptidic acylguanidine inhibitors of Cathepsin D, Pepstatin A,Bm-Aspin, SIPI, Via, RNAi-Rab27A and Solanum lycopersicum asparticprotease inhibitor (SLAPI).

Examples of Cathepsin G Inhibitors include, but are not limited to,WFDC12, Phenylmethylsulfonyl fluoride (PMSF), Ecotin, SerpinBl,SerpinA3, CeEI, or Caesalpinia echinata elastase inhibitor, SLPI(secretory leukocyte protease inhibitor), Alpha1-Antitrypsin (AAT),Bauhinia bauhinoides cruzipain inhibitor, Alpha-Aminoalkylphosphonatediaryl esters, Greglin,[2-[3-[[(1-benzoyl-4-piperidinyl)methylamino]carbonyl]-2-naphthalenyl]-1-(1-naphthalenyl)-2-oxoethyl]-phosphonicacid (KPA), Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI),Trappin-2 A62L, SV-66, SCGI, Bortezomib, Human monocyte/neutrophilelastase inhibitor (MNEI), a 42-kDa serpin protein andAnti-leukoproteinase (ALP).

Examples of Calcium Channel Blockers include, but are not limited to,Dihydropyridine Calcium Channel Blockers, Phenylalkylamine CalciumChannel Blockers, Benzothiazepine Calcium Channel Blockers,Non-Selective Calcium Channel Blockers, as well as “Other” CalciumChannel blockers.

Examples of Dihydropyridine Calcium Channel Blockers include, but arenot limited to, Amlodipine (Norvasc), Aranidipine (Sapresta),Azelnidipine (Calblock), Barnidipine (HypoCa), Benidipine (Coniel),Cilnidipine (Atelec, Cinalong, Siscard) Not available in US, Clevidipine(Cleviprex), Isradipine (DynaCirc, Prescal), Efonidipine (Landel),Felodipine (Plendil), Lacidipine (Motens, Lacipil), Lercanidipine(Zanidip), Manidipine (Calslot, Madipine), Nicardipine (Cardene, CardenSR), Nifedipine (Procardia, Adalat), Nilvadipine (Nivadil), Nimodipine(Nimotop), Nisoldipine (Baymycard, Sular, Syscor), Nitrendipine (Cardif,Nitrepin, Baylotensin), Pranidipine (Acalas).

Examples of Phenylalkylamine Calcium Channel Blockers include, but arenot limited to, Verapamil (Calan, Isoptin), Gallopamil and Fendiline.

Examples of Benzothiazepine Calcium Channel Blockers include, but arenot limited to, Diltiazem (Cardizem) and Fendiline.

Examples of Non-Selective Calcium Channel Blockers include, but are notlimited to, Mibefradil, Bepridil, Flunarizine, Fluspirilene andFendiline.

Examples of other Calcium Channel Blockers include, but are not limitedto, Gabapentin, Pregabalin and Ziconotide.

An example of DRIs includes, but is not limited to, Aliskiren.

In certain examples, the cancer stem cells may be partiallydifferentiated and committed toward a specific cell lineage associatedwith a particular tumour.

In one example, the partially differentiated cancer stem cells arecharacterised by expression of one or more tumourigenic biomarkers, orco-express with one of more tumourigenic biomarkers, selected from thegroup consisting of epithelial cancer cell markers, lymphatic cellmarkers, blood vascular markers, myeloid cell markers, as well ascombinations thereof.

Examples of epithelial cancer cell markers include, but are not limitedto, p63, epithelial membrane antigen (EMA) and cytokeratins includingCYK 5, CYK6, CYK 8 and CYK18.

Examples of cancer stem cell markers include, but are not limited to,CD44, CD133, CD24, and ALDH1.

Examples of lymphatic cell markers include, but are not limited to,LYVE-1 and VEGFR-3.

Examples of blood vascular markers include, but are not limited to, CD34and ACE.

Examples of haemogenic endothelial markers include, but are not limitedto, TAL-1.

Examples of myeloid markers include, but are not limited to, tryptaseand CD163.

Examples of epithelial to mesenchymal transition (EMT) markers include,but are not limited to, Twist, Slug, SNAIL, Bmi1 and MMP-9.

Examples of proliferation markers include, but are not limited to, Ki67.

For example, any given tumour may possess different populations ofcells, including cancer stem cells, partially differentiated cancer stemcells and mature tumour cells etc.

By way of illustration only, the partially differentiated stem cellsassociated with a squamous cell carcinoma may express certain markerssuch as EMA and p63. Similarly, partially differentiated cancer stemcells associated with leukemia may express markers such as TAL-1 andGATA-2.

As such, the present invention also contemplates not only identificationof cancer stem cells (expressing e.g., embryonic stem cells markers),but also to the identification of partially differentiated cancer stemcells that are committed to a certain cell lineage associated with aparticular tumour/cancer or mature tumour cells. Accordingly, thepresent invention may be used to provide an initial prognosis ordiagnosis as to the existence of progenitor cancer stem cells (andtherefore likelihood of possessing, or being predisposed to, cancer),which may be followed up with subsequent interrogations to determine thetype of cancer by investigating e.g., the profile of partiallydifferentiated cancer stem cells or mature tumour cells. The latterinvestigations may be conducted using biomarker expression profiles oncells obtained from a biological sample obtained from, or known to beassociated with, a tumour e.g., tissue biopsy or a blood/serum samplethrough tumour shedding. The specific cell marker signatures associatedwith partially differentiated cancer stem cells and/or mature tumourcells for any given tumour type would be well known to a person skilledin the art.

Gene expression profiles and signatures associated with cancer stemcells, for example solid tumour stem cells, as well as novel stem cellcancer markers are useful for the diagnosis, characterization, prognosisand treatment of tumours involving cancer stem cells. Biomarkerexpression analysis can be used to determine the population (orsub-populations) of cancer stem cells responsible for tumourigenesis.These sub/populations of cancer stem cells can then be targeted usingeffective treatment regimes in order to prevent the growth and spread ofa tumour, as well as its potential metastasis.

As such, the present invention is also useful in the prognosis anddiagnosis of cancer, in particular by profiling a biological fluidsample for the presence of a cancer stem cell or cancer stem cellpopulation.

Accordingly, in another aspect of the present invention there isprovided a method for determining presence or absence of cancer in asubject, the method comprising:

(i) detecting and/or measuring the level of a cancer stem cellpopulation in a biological sample obtained from the subject usingbiomarker expression analysis;

(ii) comparing the level of the cancer stem cell population obtainedfrom the sample against the cancer stem cell level from a controlpopulation, wherein, an increased level in the cancer stem cellpopulation obtained from the biological sample relative to the controlpopulation is diagnostic that the subject has, or is predisposed todeveloping, cancer. In another aspect of the present invention there isprovided a method for determining presence or absence of cancer in asubject, the method comprising:

(i) detecting and/or measuring the level of a cancer stem cellpopulation in a biological sample obtained from the subject usingbiomarker expression analysis;

(ii) comparing the level of the cancer stem cell population obtainedfrom the sample against the cancer stem cell level from a controlpopulation,

wherein, an increased level in the cancer stem cell population obtainedfrom the biological sample relative to the control population isdiagnostic that the subject has, or is predisposed to developing,cancer, and

(iii) administering a prophylactic or therapeutic regime to the subjectwho has, or is predisposed to developing, cancer.

In certain examples according to the prognostic and diagnostic methodsof the present invention, the cancer stem cells are characterised byexpression of one or more embryonic stem cell biomarkers as well asexpression of one or more biomarkers associated with theRenin-Angiotensin System. In one example, the one or more embryonic stemcell markers is selected from the group consisting of OCT4, SOX2, NANOG,CD44, SAL-4, SSEA4, PSTAT3, and H1F1. In another example, the one ormore embryonic stem cell biomarkers consists in OCT4, SOX2, NANOG andPSTAT3. In yet another example, the one or more biomarkers associatedwith the Renin-Angiotensin System is selected from the group consistingof RR, ATIIR2 and sRR.

In other examples according to the prognostic and diagnostic methods ofthe present invention, the therapeutic agent is selected from the groupconsisting of ACEIs, ARBs, Direct DRIs, Beta-Blockers, Cyclo-oxygenase 2Inhibitors, Chymase Inhibitors, Inhibitors of Cathepsin B, Cathepsin Dand Cathepsin G, Calcium Channel Blockers, Calcium Supplements andVitamin D.

In another aspect of the present invention there is provided a methodfor determining presence or absence of cancer in a subject, the methodcomprising:

(i) detecting and/or measuring the level of a partially differentiatedcancer stem cell population in a biological sample obtained from thesubject using biomarker expression analysis;

(ii) comparing the level of the partially differentiated cancer stemcell population obtained from the sample against the partiallydifferentiated cancer stem cell level from a control population,

wherein, an increased level in the partially differentiated cancer stemcell population obtained from the biological sample relative to thecontrol population is diagnostic that the subject has, or is predisposedto developing, cancer.

In another aspect of the present invention there is provided a methodfor determining presence or absence of cancer in a subject, the methodcomprising:

(i) detecting and/or measuring the level of a partially differentiatedcancer stem cell population in a biological sample obtained from thesubject using biomarker expression analysis;

(ii) comparing the level of the partially differentiated cancer stemcell population obtained from the sample against the partiallydifferentiated cancer stem cell level from a control population,

wherein, an increased level in the partially differentiated cancer stemcell population obtained from the biological sample relative to thecontrol population is diagnostic that the subject has, or is predisposedto developing, cancer, and

(iii) administering a prophylactic or therapeutic regime to the subjectwho has, or is predisposed to developing, cancer.

In an example according to the prognostic and diagnostic methods of thepresent invention, the biomarker analysis is performed using geneexpression and/or protein analysis techniques.

The therapeutic agent(s) comprises any known or as yet unidentifiedtherapeutic which selectively targets the cancer stem cell population.Examples of known therapeutics are listed herein. In an example, thetherapeutic agent results in an approximately 25% reduction, anapproximately 40% reduction, an approximately 50% reduction or anapproximately 75% reduction in cancer cells. This includes both cancerstem cells and mature/bulk tumour cells. In a related example, reductionin cancer cells is determined by comparing the amount of cells with acancer cell marker phenotype present in a tissue sample from the humansubject to the amount of cells with the same cancer cell markerphenotype present in a tissue sample from the same human subject at anearlier time point.

The growth, proliferation and/or differentiation of cancer stem cellsmay be selectively eradicated or inhibited using a therapeutic agent.Accordingly, in one example, growth, proliferation and/ordifferentiation of the cancer stem cells is selectively inhibited usinga therapeutic agent that binds to a cell surface antigen and arrests thegrowth and proliferation of the cancer stem cells.

In another example, growth, proliferation and/or differentiation of thecancer stem cells is selectively inhibited using a therapeutic agentthat comprises a differentiation factor capable of differentiating acancer stem cell to a non-malignant cell lineage or away from amalignant cell lineage potential. The differentiated cells no longerretain the ability to be tumourigenic.

By effectively arresting the growth, proliferation and/ordifferentiation of the cancer stem cells, their ability to differentiateinto tumour/bulk cells is also inhibited, resulting in reduced spreadand growth of the tumour.

The inventors of the present application have also surprisinglyidentified that the cancer stem cells co-express with lymphatic celllineages (e.g. lymphatic cells which express, for example LYVE-1 and/orVEGFR-3). Accordingly, this would suggest that the spread and metastasisof cancer may be via the lymphatic system rather than the more acceptedblood vasculature (i.e., angiogenesis) and provides alternative targetsas a means for preventing the spread of cancer to secondary sites (i.e.,metastasis).

In certain examples, the therapeutic agent results in the stabilisationof a cancer stem cell population after a period of time (e.g., after 2,5, 10, 20, 30 or more doses of a therapy, or after 2 weeks, 1 month, 2months, 1 year, 2 years, 3 years, 4 years or more). In other examples,the therapeutic agent achieves a 5%-40%, 10%-60%, 20% to 99% or higherreduction in the cancer stem cell population. In other examples, areduction in a cancer stem cell population is achieved after 2 weeks, 1month, 2 months, 3 months, 4 months, 6 month, 9 months, 1 year, 2 years,3 years, or 4 years of administration of one or more therapies. Infurther examples, in accordance with the therapy, the reduction in acancer stem cell population is monitored periodically (e.g., after 2, 5,10, 20, 30 or more doses of one or more therapies, or after 2 weeks, 1month, 2 months, 1 year, 2 years, 3 years, 4 years or more afterreceiving one or more therapies). The stabilization, reduction orelimination of a cancer stem cell population stabilises, reduces oreliminates the cancer cell population produced by the cancer stem cellpopulation, and thus, stabilises, reduces or eliminates the growth of atumour, the bulk size of a tumour, the formation of a tumour and/or theformation of metastases. In other words, the stabilization, reduction orelimination of the cancer stem cell population prevents the formation,reformation or growth of a tumour and/or metastases by cancer cells.

Therapeutic agent or agents according to the present invention caninhibit cancer stem cell growth in vitro and in vivo. Examples ofdifferent therapeutic agents are listed in further detail below.

Conventional cancer therapies, including chemotherapy, target maturetumour cells in an attempt to control or treat the cancer. For example,conventional chemotherapy agents seek to interrupt normal cell-cycle. Asa consequence, standard chemotherapy only effectively targets thosecells that are rapid-cycling. Whereas mature tumour cells arerapid-cycling, cancer stem cells are slow-cycling. As such, conventionalchemotherapy involves the targeting and killing of mature tumour cells,as well as other non-tumour rapid-cycling cells present, but having anenriching effect on cancer stem cells, as they are not targeted and socontinue to proliferate.

This then gives the cancer stem cells an opportunity to eitherrepopulate the tumour bulk cells, resulting in tumour recurrence, ormetastasise and form a secondary tumour elsewhere in the body.

Cancer stem cell biology is still poorly understood, with many studiesfocusing on identifying single markers that can be used as independentprognostic indicators and identifiers of these cells.

The present inventors have investigated the overall protein expressionprofile of cancer stem cells and ultimately the molecular systems thatcontrol tumourigenesis. For example, it is likely that the RAS may playa role in this (supported by the expression of ACE by these cells; referto Example 2/Table 5), although other important molecular triggers andsignalling pathways are also likely to be involved.

By understanding the molecular triggers and signalling pathways involvedin tumourigenesis, with an emphasis on the role of cancer stem cellpopulations, the inventors have identified novel treatment targets thatwill allow selective targeting of the cancer stem cells, therebyeliminating the driving force behind cancer. In addition, by inducingdifferentiation of these cells to a mature cell type such as adiposetissue, this would eradicate the malignant potential of these cells.

In addition to the methods described herein, the present invention alsoprovides pharmaceutical compositions useful in the treatment orprevention of cancer. The pharmaceutical compositions of the presentinvention comprise therapeutic agents sufficient to selectivelyeradicate, or inhibit the growth, proliferation and/or differentiationof cancer stem cells in a tumour associated with a cancer.

Accordingly in another aspect of the present invention there is provideda pharmaceutical composition for use in a method for treatment ofcancer, wherein the pharmaceutical composition comprises a therapeuticagent(s) sufficient to selectively inhibit eradicate, or the growth,proliferation and/or differentiation of cancer stem cells in a tumourassociated with a cancer, and wherein the method comprises administeringthe therapeutic agent to a patient with cancer.

In yet another aspect of the present invention there is provided a kitor article of manufacture for use in the treatment of cancer, the kitcomprising a therapeutic agent(s) sufficient to selectively eradicate,or inhibit the growth, proliferation and/or differentiation of cancerstem cells in a tumour associated with a cancer, together withinstructions for how to administer a therapeutic dose to the subject.

Since the cancer stem cells according to the present invention have beenshown to express key regulatory components of the (e.g.)Renin-Angiotensin System and/or the Pro/Renin System, the cancer stemcell populations associated with certain tumours may be effectivelytargeted by administering a therapeutic amount of a drug which is knownto also target these systems. Examples include, but are not limited to,Angiotensin-Converting Enzyme Inhibitors (ACEIs), Angiotensin ReceptorBlockers (ARBs), Direct Renin Inhibitors (DRIs), Beta-Blockers,Cyclo-oxygenase 2 Inhibitors, Chymase Inhibitors, Cathepsin Inhibitorsincluding Cathepsin B Inhibitors, Cathepsin D Inhibitors and Cathepsin GInhibitors, Calcium Channel Blockers, Calcium Supplements and Vitamin D,as described above.

In certain examples the method for treatment of cancer comprisesdetermining in vitro that the therapeutic agent can decrease the amountof cancer stem cells in a sample comprising cancer stem cells, prior toadministration of the therapeutic agent.

Further detail with respect to the present invention is presented underthe following sub-headings.

Renin-Angiotensin System

The renin-angiotensin system (RAS) is traditionally known to preservefluid volume during periods of restricted dietary salt and also preventsischaemia during acute volume loss. The main effector peptide of the RASis angiotensin II (ATII). It induces vasoconstriction and sympatheticactivation, raises aldosterone levels, and promotes renal salt and waterretention via the angiotensin II receptor 1 (ATIIR1). Over the last fewdecades, the RAS has been a drug target of particular interest becauseof its involvement in cardiovascular disease (CVD) and renovasculardisease. The CVD and renovascular disease can be understood as acontinuum of risk factors, target organ damage, events, and mortality.Risk factors (such as hypertension, dyslipidemia, diabetes, and smoking)led to the development of target organ damage including atherosclerosis,left ventricular hypertrophy (LVH), and renal impairment. Target organdamage progressively worsens, leading ultimately to myocardialinfarction (MI), heart failure (HF), end-stage renal disease (ESRD),stroke, or death.

ATII the main effector peptide of the RAS, plays an active role duringall stages of this continuum. The first step in the RAS cascade is theformation of angiotensin I (ATI) from the precursor angiotensinogenunder the action of renin; early evidence for the importance of RAS inCVD came from the consistent finding that renin activity is predictiveof the risk of cardiovascular (CV) events. ATI is then converted toATII, the principal effector peptide of the RAS, byangiotensin-converting enzyme (ACE). In addition, ATII can be producedin tissues by enzymes such as chymase. This locally produced ATII isbelieved to mediate paracrine and autocrine functions. ATII acts viaATIIR1 and ATIIR2. Activation of ATIIR1 results in vasoconstriction,aldosterone and vasopressin secretion, sodium retention, and decreasedrenal perfusion. Hence, these receptors mediate the deleterious effectsof ATII, including elevated blood pressure (BP) and cardiac and vascularremodelling. The effects of the ATII receptors have been less clearlydefined because of the limited expression of these receptors in adults,because of their unconventional signaling pathways, and because manyATII-mediated actions are masked by opposing ATI-mediated effects.However, it is now recognised that ATIIR2 generally oppose the actionsof ATIIR1, mediating various antiproliferative and anti-inflammatoryeffects and promoting tissue differentiation and regeneration andapoptosis.

Additional components of the RAS have been identified in the lastdecade, including bioactive angiotensin peptides, such as angiotensinIII, angiotensin IV, and angiotensin-(1-7), the effects of which havenot yet been fully elucidated for the CV and renal system.

The discovery of the renin receptor has shed further light on thebiology of the RAS. Renin, simply considered until recently as therate-limiting enzyme of RAS activation, has also turned out to be theligand for a protein known as the renin/prorenin receptor that bindsrenin and prorenin about equally, regardless of their biologicactivities. Prorenin, which represents 70% to 90% of total circulatingrenin, when bound to the receptor induces an increase in the catalyticefficiency of angiotensinogen conversion to ATI, which contributes tolocal production of ATII and its systemic levels, as well as binding ofrenin/prorenin to the renin/prorenin receptor, exerting physiologiceffects that are independent of ATII, including activation ofintracellular signal pathways, enhanced synthesis of DNA, andstimulation of the release of plasminogen activator inhibitor 1,collagen 1, fibronectin, and transforming growth factor β-1.6

There are a number of known drugs which target the RAS. The two majorclasses of drugs that target the RAS are the angiotensin-convertingenzyme (ACE) inhibitors and the selective ATI receptor blockers (ARBs).Although both of these drug classes target ATII, the differences intheir mechanisms of action have implications for their effects on otherpathways and receptors that may have therapeutic implications. BothACEIs and ARBs are effective antihypertensive agents that have beenshown to reduce the risk of cardiovascular and renal events.

Direct inhibition of renin, the most proximal aspect of the RAS, becameclinically feasible from 2007 with the introduction of Aliskiren. Thislatter drug has been shown to be efficacious for the management ofhypertension. Combined therapy of direct renin-inhibitors with ACEIs orARBs has been tested in some clinical situations as congestive HF andproteinuria with diverse results.

RAS drugs include, but are not limited to, Angiotensin-Converting EnzymeInhibitors (ACEIs), Angiotensin Receptor Blockers (ARBs), Direct ReninInhibitors (DRIs), Beta-Blockers, Cyclo-oxygenase 2 Inhibitors, ChymaseInhibitors, Cathepsin Inhibitors including Cathepsin B Inhibitors,Cathepsin D Inhibitors and Cathepsin G Inhibitors, Calcium ChannelBlockers, Calcium Supplements and Vitamin D, as described above.

Squamous Cell Carcinomas

Squamous cells are flat cells that form the surface layers of anepithelium. They can be identified histologically by the fact that theylook flattened and thin under a microscope. Epithelia lined by squamouscells can be classified as either simple squamous epithelium orstratified squamous epithelium.

Squamous cell carcinomas refers to the epithelial tumours found in manydifferent organs, including the skin, upper aerodigestive tractincluding the coral cavity, nasal cavity, oesophagus, lungs, cervix andgastrointestinal tract, which show squamous cell differentiation.Included are head and neck squamous cell carcinomas, lung squamous cellcarcinomas, skin squamous cell carcinomas, otic squamous cellcarcinomas, vulval squamous cell carcinomas, cervical squamous cellcarcinomas, oesophageal squamous cell carcinomas, and the like. It is amalignant tumour of the epithelium that shows squamous celldifferentiation. Squamous cell carcinoma is usually developed in theepithelial layer of the skin and sometimes in various mucous membranesof the body. This type of cancer can be seen on the skin, lips, insidethe mouth, larynx or oesophagus.

The most common non-cutaneous tumour of the head and neck is squamouscell carcinoma of the of the upper aerodigestive tract (includinglarynx, oropharynx, oral cavity [including oral tongue and floor of themouth]). Somewhat less common are tumours of the salivary gland, jaw,nose and paranasal sinuses, and ear.

Most head and neck cancers first manifest as an asymptomatic lump,ulceration, or visible mucosal lesion (e.g., leukoplakia,erythroplakia). Subsequent symptoms depend on location and extent of thetumour and include pain, paresthesia, nerve palsies, trismus, andhalitosis. Head and neck cancers may remain localised for months toyears. Local tissue invasion is eventually followed by metastasis toregional lymph nodes. Distant lymphatic metastases tend to occur late.Haematogenous metastases are usually associated with large or persistenttumours and occur more commonly in immunocompromised patients. Commonsites of distant metastases are the lungs, liver, bone, and brain.

Oral cavity and oropharyngeal squamous cell carcinoma affects about30,000 Americans each year. Oral squamous cell carcinoma is the mostcommon oral cavity and oropharyngeal cancer. The chief risk factors fororal cavity squamous cell carcinoma are smoking and/or alcohol use.Squamous cell carcinoma of the oral tongue may also result, fromPlummer-Vinson syndrome, syphilis, or chronic trauma. About 40% of oralcavity squamous cell carcinomas affect the oral tongue and 20% affectthe floor of the mouth, with the remainder affecting the lip buccalmucosal, retromolar trigone, alveolus and hard palate. Oropharyngealsquamous cell carcinoma affects the base of the tongue and soft palateand tonsillar area and have been associated human papilloma virusinfection with alcohol and smoking playing a lesser role.

Oral lesions are asymptomatic initially. They may appear in areas oferythroplakia or leukoplakia and may be exophytic or ulcerated. Bothvariants are indurated and firm with a rolled border. Oraopharyngealcancer usually presents as an asymmetric swelling and sore throat; painoften radiates to the ipsilateral ear. A metastatic mass in the neck maybe the first symptom.

If squamous cell carcinoma of the tongue is localised (no lymph nodeinvolvement), 5-yr survival is about 50%. For localised squamous cellcarcinoma of the floor of the mouth, 5-yr survival is 65% but with lymphnode metastasis, the 5-yr survival is 20%. For lower lip lesions, 5-yrsurvival is 90%, and metastases are rare. Squamous cell carcinoma of theupper lip tends to be more aggressive and metastatic. For oropharyngealsquamous cell carcinoma, 5-yr survival is 68% if patients are treatedbefore lymph node involvement but only 17% after involvement. Metastasesreach the regional lymph nodes first and later the lungs. Surgery andradiation therapy are the treatments of choice for oral cavity cancer.Radiotherapy and often chemotherapy are the treatment of choice fororopharyngeal cabcer

Characterization of Squamous Carcinoma Stem Cells

In squamous cell carcinomas, characterisation of cancer stem cellsallows for the development of new treatments that are specificallytargeted against this critical population of cells, particularly theirability to self-renew, resulting in more effective therapies.

In human squamous cell carcinomas it is shown herein that there is asubpopulation of tumourigenic cancer cells with both self-renewal anddifferentiation capacity. These tumourigenic cells are responsible fortumour maintenance, and also give rise to large numbers of abnormallydifferentiating progeny that are not tumourigenic, thus meeting thecriteria of cancer stem cells.

Cancer stem cells of squamous carcinoma are identified by theirphenotype with respect to particular markers, and/or by their functionalphenotype. In some examples, the these cancer stem cells are identifiedand/or isolated by binding to the cell with reagents specific for themarkers of interest. The cells to be analysed may be viable cells, ormay be fixed or embedded cells.

In some examples, the reagents specific for the markers of interest areantibodies, which may be directly or indirectly labeled. Such antibodieswill usually include antibodies specific for CD44 and antibodiesspecific for a lineage panel. The lineage panel will usually includereagents specific for markers of normal leukocytes, fibroblasts,endothelial, mesothelial cells, etc. Such markers may include reagentsspecific for one or more, two or more, three or more of the followingmarkers: CD44, SOX2, OCT4, NANOG, and a lack of expression of p63, CD34and EMA.

Cancer Therapy—General

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, or is currently being used for the prevention, treatment,and/or management of cancer can be used in compositions and methods ofthe present invention. Therapies (e.g., therapeutic or prophylacticagents) include, but are not limited to, peptides, polypeptides,antibodies, conjugates, nucleic acid molecules, small molecules, mimeticagents, synthetic drugs, inorganic molecules, and organic molecules.Non-limiting examples of cancer therapies include chemotherapies,radiation therapies, radioimmunotherapies, hormonal therapies, targetedtherapies, epigenetic therapies, differentiation therapies,anti-angiogenic therapies small molecule therapies, epigenetictherapies, toxin therapies, differentiation therapies, prodrugactivating enzyme therapies, antibody therapies, protein therapies,and/or biological therapies including immunotherapies, and surgery. Incertain examples, a prophylactically and/or therapeutically effectiveregimen of the invention comprises the administration of a combinationof therapies.

Examples of cancer therapies include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carrnustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;gemcitabine hydrochloride; histone deacetylase inhibitors (HDAC-Is);hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; imatinibmesylate (Gleevec, Glivec); interleukin II (including recombinantinterleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b;interferon alpha-nI; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; lenalidomide (Revlimid); letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantronehydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride;anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; InternationalPublication No. WO 02/098370, which is incorporated herein by referencein its entirety)); megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxaliplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; /-/; antagonist D; antagonistG; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-D L-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; InternationalPublication No. WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metal loproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumour suppressor 1-based therapy; mustardanticancer agent; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides;onapristone; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; gammasecretase inhibitors, single chain antigen binding protein; sizofiran;sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol;somatomedin binding protein; sonermin; sparfosic acid; spicamycin D;spiromustine; splenopentin; spongistatin 1; squalamine; stem cellinhibitor; stem-cell division inhibitors; stipiamide; stromelysininhibitors; sulfinosine; superactive vasoactive intestinal peptideantagonist; suradista; suramin; swainsonine; syntheticglycosaminoglycans; tallimustine; 5-fluorouracil; leucovorin; tamoxifenmethiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide;teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine;thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone;tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;toremifene; totipotent stem cell factor; translation inhibitors;tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin;tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBCinhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor;urokinase receptor antagonists; vapreotide; variolin B; vector system,erythrocyte gene therapy; thalidomide; velaresol; veramine; verdins;verteporfin; vinorelbine; vinxaltine; anti-integrin antibodies (e.g.,anti-integrin avp3 antibodies); vorozole; zanoterone; zeniplatin;zilascorb; and zinostatin stimalamer. A non-limiting list of compoundsthat could be used to target cancer stem cells includes: inhibitors ofinterleukin-3 receptor (IL-3R) and CD123 (including peptides,peptide-conjugates, antibodies, antibody-conjugates, antibody fragments,and antibody fragment-conjugates that target IL-3R or CDl 23);cantharidin; norcantharidin and analogs and derivatives thereof; Notchpathway inhibitors including gamma secretase inhibitors; sonichedgehog/smoothened pathway inhibitors including cyclopamine and analogsthereof; antibodies to CD96; certain NF-kB/proteasome inhibitorsincluding parthenolide and analogs thereof; certain triterpenesincluding celastrol; certain mTOR inhibitors; compounds and antibodiesthat target the urokinase receptor; sinefungin; certain inosinemonophosphate dehydrogenase (IMPDH) inhibitors; PPAR-alpha andPPAR-gamma agonists and antagonists (including pioglitazone,tesaslitazar, muraglitazar, peliglitazar, lobeglitazone, balaglitazone,ragaglitazar, rosiglitazone, farglitazar, sodelglitazar, reglitazar,naveglitazar, oxeglitazar, metaglidasen, netoglitazone, darglitazone,englitazone, thiazolidinediones, aleglitazar, edaglitazone,rivoglitazone, troglitazone, imiglitazar, and sipoglitazar); telomeraseinhibitors; antibodies to EpCAM (ESA); GSK-3 beta agonists andantagonists (including Lithium, 6-bromoinirubin-3′-oxime (BIO), TDZD8);Wnt pathway inhibitors including antibodies to frizzled or smallmolecules that inhibit disheveled/frizzled or beta catenin; anti-CD20antibodies and conjugates (e.g. Rituxan, Bexxar. Zevalin) for novel usein multiple myeloma or melanoma; anti-CD133 antibody; anti-CD44antibody; antibodies to IL-4; certain differentiation agents such asversnarinone; compounds that target CD33 such as an antibody orbetulinic acid; compounds that target lactadherin such as an antibody;small molecules or antibodies that target CXCR4 or SDF-I; smallmolecules or antibodies that target multi-drug resistance pumps;inhibitors of survivin; inhibitors of XIAP; small molecules that targetBcI-2; antibodies to CLL-I; and furin inhibitors (such ascucurbitacins). An additional non-limiting list of compounds that couldalso be used to target cancer stem cells includes i) antibodies,antibody fragments, and proteins that are either naked or conjugated toa therapeutic moiety that target certain cell surface targets on cancerstem cells, or ii) small molecules known in the art including ones thatcan be further optimized (e.g. via chemistry) or identified via a cancerstem cell-based screen (e.g. such as one that would determine whether acompound impairs proliferation or viability of a cancer stem cellthrough standard methods, the cell surface and intracellular targetsincluding (not meant to be exhaustive) are: Rexl (Zfp42), CTGF, ActivinA, Wnt, FGF-2, HIF-I, AP-2gamma, Bmi-1, nucleostemin, hiwi, Moz-TIF2,Nanog, beta-arrestin-2, OCT4, SOX2, stella, GDF3, RUNX3, EBAF, TDGF-I,nodal, ZFPY, PTNE, EvM5 Pax3, McI-I, c-kit, Lex-1, Zfx, lactadherin,aldehyde dehydrogenase, BCRP, telomerase, CD133, Bcl-2, CD26, Gremlin,and FoxC2.

In some examples, the therapy(ies) used is an immunomodulatory agent.Non-limiting examples of immunomodulatory agents include proteinaceousagents such as cytokines, peptide mimetics, and antibodies (e.g., human,humanised, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)₂fragments or epitope binding fragments), nucleic acid molecules (e.g.,antisense nucleic acid molecules and triple helices), small molecules,organic compounds, and inorganic compounds. In particular,immunomodulatory agents include, but are not limited to, methotrexate,leflunomide, cyclophosphamide, Cytoxan, Immuran. cyclosporine A,minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)),methylprednisolone (MP), corticosteroids, steroids, mycophenolatemofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamides (e.g., lefiunamide), T cell receptor modulators,cytokine receptor modulators, and mast cell modulators. Other examplesof immunomodulatory agents can be found, e.g., in U.S. Publication No.2005/0002934 A1 at paragraphs 259-275 which is incorporated herein byreference in its entirety. In one example, the immunomodulatory agent isa chemotherapeutic agent. In an alternative example, theimmunomodulatory agent is an immunomodulatory agent other than achemotherapeutic agent. In some examples, the therapy(ies) used inaccordance with the invention is not an immunomodulatory agent.

In some examples, the therapy(ies) used is an anti-angiogenic agent.Non-limiting examples of anti-angiogenic agents include proteins,polypeptides, peptides, fusion proteins, antibodies (e.g., human,humanised, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments,F(ab)₂ fragments, and antigen-binding fragments thereof) such asantibodies that specifically bind to TNF-α, nucleic acid molecules(e.g., antisense molecules or triple helices), organic molecules,inorganic molecules, and small molecules that reduce or inhibitangiogenesis. Other examples of anti-angiogenic agents can be found,e.g., in U.S. Publication No. 2005/0002934 A1 at paragraphs 277-282,which is incorporated by reference in its entirety. In other examples,the therapy(ies) used in accordance with the invention is not ananti-angiogenic agent.

In certain examples, the therapy used is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, cholorambucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, mephalen,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclins include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etopiside (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine).

The invention includes the use of agents that target cancer stem cells.In certain examples, the agent is a small molecule, biologic, or anagent including a peptide or antibody or antibody fragment that is nakedor is attached directly or indirectly to a therapeutic moiety viachemical or recombinant technology. Non-limiting examples of therapeuticmoieties include, but are not limited to, therapeutic enzymes,chemotherapeutic agents, cytokines, bacterial toxins, diphtheria toxin,Pseudomonas exotoxin, radionuclides, RNase, and antimetabolites. In someexamples, the agent used is an agent that binds to a marker, e.g., anantigen on a cancer stem cell. In a specific example, the agent binds toan antigen that is expressed at a greater level on cancer stem cellsthan on normal stem cells. In another specific example the agent bindsto an antigen that is expressed at the same level on cancer stem cellsas on normal stem cells.

In a specific example, the agent binds specifically to a cancer stemcell antigen that is not, or is, on a normal stem cell. In otherexamples, the therapy(ies) used in accordance with the invention is anagent that binds to a marker on cancer stem cells, In one example, theagent that binds to a marker on cancer stem cells is an antibody orantibody fragment—either of which may be naked or conjugated to atherapeutic moiety such as therapeutic enzymes, chemotherapeutic agents,cytokines, bacterial toxins, diphtheria toxin, Pseudomonas exotoxin,radionuclides, RNase, and antimetabolites.

For example, in a specific example, the agent binds specifically to theIL-3 Receptor (IL-3R) or the α-subunit thereof (i.e., the CD123antigen). In some examples, the agent that binds to the IL-3R is anantibody that is specific for IL-3R or the α-subunit thereof. Theantibody may be conjugated to a therapeutic moiety (e.g., achemotherapeutic agent, a plant-, fungus- or bacteria-derived toxin, ora radionuclide, RNase) using a linking agent, either chemically orrecombinantly, to effect a cell killing response. In certain examples,the antibody or antibody-conjugate binds to the α-subunit of IL-3R(i.e., the CDl 23 antigen). In other words, the antibody orantibody-conjugate binds to the IL-3R α-subunit but not the IL-3Rβ-subunit. In other examples, the antibody or antibody-conjugateimmuospecifically binds to the IL-3R, containing both the α and βsubunits. Methods for preparing antibodies to IL-3R and mimetics ofantibodies to IL-3R are described, e.g., in U.S. Pat. No. 6,733,743 B2,which is incorporated herein by reference in its entirety.

In other examples, the agent that binds to a marker on cancer stem cellsis a ligand. In some examples, the ligand is a cytokine that binds to acytokine receptor on cancer stem cells. In a particular example, theligand is interleukin-3 (IL-3) which can be conjugated to a therapeuticmoiety including a toxin. The IL-3-toxin conjugate can be in the form ofa fusion protein in examples where the toxin is a protein, such asdiphtheria toxin. Methods for preparing and isolating an IL-3-diphtheriatoxin fusion protein (“IL3DT”) are described in Frankel et al.,“Diphtheria toxin fused to human interleukin-3 is toxic to blasts frompatients with myeloid leukemias,” Leukemia 14:576 (2000) and Urieto etal., “Expression and purification of the recombinant diphtheria fusiontoxin DT388IL3 for phase I clinical trials,” Protein Expression andPurification 33: 123-133 (2004),‘the disclosures of which areincorporated by reference in their entireties. In other examples, thetherapy is not IL3DT.

In certain examples, antibodies that bind to a marker on cancer stemcells are substantially non-immunogenic in the treated subject. Methodsfor obtaining non-immunogenic antibodies include, but are not limitedto, chimerizing the antibody, humanising the antibody, generatingantibody fragments, and generating antibodies from the same species asthe subject receiving the therapy. See, for example, paragraphs 539-573of U.S. Publication No. 2005/0002934 A1, which is incorporated byreference in its entirety. Antibodies that bind to markers in cancerstem cells can be produced using techniques known in the art.

In some examples, the therapy used comprises the use of x-rays, gammarays and other sources of radiation to destroy cancer stem cells and/orcancer cells. In specific examples, the radiation therapy isadministered as external beam radiation or teletherapy, wherein theradiation is directed from a remote source. In other examples, theradiation therapy is administered as internal therapy or brachytherapywherein a radioactive source is placed inside the body close to cancerstem cells, cancer cells and/or a tumour mass.

In some examples, the therapy used is a proliferation based therapy.Non-limiting examples of such therapies include a chemotherapy andradiation therapy as described supra.

Currently available therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006). Routes of administration known in the art include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal,ocular, intradermal, intratumoural, intracerebral, intrathecal, andintranasal. In some examples, the therapies are administered as part ofa composition comprising a pharmaceutically acceptable carrier orexcipient.

In Vitro Assays

The therapies described herein can be tested in vitro and/or in vivo fortheir ability to reduce the amount of cancer cells and/or cancer cells,or inhibit their proliferation. The ability of a therapy to stabilize orreduce the amount of cancer stem cells, cancer cells and/or immune cells(e.g., lymphocytes) or inhibit their proliferation can be assessed by:detecting the expression of antigens on cancer stem cells, cancer cells,and immune cells; detecting the proliferation cancer stem cells, cancercells and immune cells; detecting the cancer stem cells and cancer cellsusing functional assays. Techniques known to those of skilled in the artcan be used for measuring these activities. For example, cellularproliferation can be assayed by ³H-thymidine incorporation assays andtrypan blue cell counts. Antigen expression can be assayed, for example,by immunoassays including, but are not limited to, competitive andnon-competitive assay systems using techniques such as Western blots,immunohistochemistry radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, immunofluorescence, flow cytometry, and FACS analysis.

A compound, pharmaceutical composition, therapeutic or prophylacticagent of the present invention may be tested in vitro and then in vivofor the desired therapeutic or prophylactic activity prior to use inhumans. For example, assays which can be used to determine whetheradministration of a specific compound is effective include cell andtissue culture assays in which a patient tissue sample (e.g., a cancercell or cancer stem cell) is grown in culture and exposed to, orotherwise contacted with, a compound, and the effect of such compoundupon the tissue sample is observed. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective therapy (e.g., prophylactic ortherapeutic agent) for each individual patient.

A therapy is preferably tested in vitro and then in vivo for the desiredtherapeutic or prophylactic activity prior to use in humans. Forexample, assays which can be used to determine whether administration ofa specific compound is effective include cell and tissue culture assaysin which a patient tissue sample (e.g., a cancer cell or cancer cell) isgrown in culture and exposed to, or otherwise contacted with, aprophylactic or therapeutic compound, and the effect of such compoundupon the tissue sample is observed. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective therapy (e.g., prophylactic ortherapeutic agent) for each individual patient. In certain examples, theeffect of a therapy is assessed in a cell viability assay using standardassays known in the art such as, for example, the XTT assay.

Toxicity Assays

The toxicity and/or efficacy of the therapies described herein can bedetermined by standard pharmaceutical procedures in cell or tissuecultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD5₀/ED₅₀-Regimens that exhibit largetherapeutic indices are preferred. While regimens that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such agents to the site of affected tissue in orderto minimise potential damage to uninfected cells and, thereby, reduceside effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the therapies for use inhumans. The dosage of such agents lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity to normal tissues. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilised. For any therapy used in the method of the invention, theprophylactically and/or therapeutically effective dose can be estimatedinitially from cell culture assays. A dose may be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels of compounds in plasma may be measured, for example, byhigh performance liquid chromatography.

Articles of Manufacture

The present invention also encompasses a finished packaged and labelledpharmaceutical product(s). This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. Thepharmaceutical product may contain, for example, a prophylactic ortherapeutic agent in a unit dosage form in a first container, and in asecond container, sterile water for injection. Alternatively, the unitdosage form may be a solid suitable for oral, transdermal, intranasal,or topical delivery.

In a specific example, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses solutions, preferably sterile, suitable foreach delivery route.

In some examples, the pharmaceutical product is a prophylactic and/ortherapeutic agent disclosed herein. In some examples, the pharmaceuticalproduct is a composition comprising a prophylactic and/or therapeuticagent and a pharmaceutically acceptable carrier or excipient. In aspecific example, the pharmaceutical composition is in a form for anappropriate route of administration. Such routes include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal,ocular, intradermal, intratumoural, intracerebral, intrathecal, andintranasal routes.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, the frequency ofadministration, the duration of administration monitoring procedures forcancer cell counts, cancer stem cell counts, lymphocyte counts,neutrophil counts, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises a prophylactic or therapeutic agent, and wherein saidpackaging material includes instruction means which indicate that saidagent can be used to prevent, manage, treat, and/or ameliorate one ormore symptoms associated with cancer, or one or more symptoms thereof byadministering specific doses and using specific dosing regimens asdescribed herein.

In certain examples, the article of manufacture include labeledantibodies that selectively or specifically bind to cancer stem cells,and that selectively or specifically bind to cancer cells. As such, thearticle contains a method to adjust the dosages used in the treatmentregimens, and to monitor the efficacy of the regimens.

The present invention provides that the adverse effects that may bereduced or avoided by the methods of the invention are indicated ininformational material enclosed in an article of manufacture for use inpreventing, treating and/or managing cancer. Adverse effects that may bereduced or avoided by the methods of the invention include, but are notlimited to, vital sign abnormalities (fever, tachycardia, bardycardia,hypertension, hypotension), haematological events (anemia, lymphopenia,leukopenia, thrombocytopenia), headache, chilis, dizziness, nausea,asthenia, back pain, chest pain (chest pressure), diarrohea, myalgia,pain, pruritus, psoriasis, rhinitis, sweating, injection site reaction,and vasodilation.

Further, the information material enclosed in an article of manufacturefor use in preventing, treating and/or managing cancer can indicate thatforeign proteins may also result in allergic reactions, includinganaphylaxis, or cytosine release syndrome. The information materialshould indicate that allergic reactions may exhibit only as mildpruritic rashes or they may be severe such as erythroderma,Stevens-Johnson syndrome, vasculitis, or anaphylaxis. The informationmaterial should also indicate that anaphylactic reactions (anaphylaxis)are serious and occasionally fatal hypersensitivity reactions. Allergicreactions including anaphylaxis may occur when any foreign protein isinjected into the body. They may range from mild manifestations such asurticaria or rash to lethal systemic reactions. Anaphylactic reactionsoccur soon after exposure, usually within 10 minutes. Patients mayexperience paresthesia, hypotension, laryngeal oedema, mental statuschanges, facial or pharyngeal angioedema, airway obstruction,bronchospasm, urticaria and pruritus, serum sickness, arthritis,allergic nephritis, glomerulonephritis, temporal arthritis, oreosinophilia.

Kits

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with reagents for detecting,monitoring and/or measuring cancer stem cells. In one example, thepharmaceutical pack or kit optionally comprises instructions for the useof the reagents provided for detecting and/or measuring cancer stemcells. In another example, the pharmaceutical pack or kit optionallycomprises a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,for use or sale for human administration.

In an example, the pharmaceutical pack or kit comprises in one or morecontainers a cancer stem cell surface marker-binding agent. In certainexamples, the agent is an antibody that selectively or specificallybinds to a cancer stem cell surface marker. The agent may be an antibody(including, e.g., human, humanised, chimeric, monoclonal, polyclonal,Fvs, ScFvs, Fab or F(ab)2 fragments or epitope binding fragments), whichcross-reacts with any cancer stem cell surface marker. In anotherexample, the antibody reacts with any one of the cancer stem cellsurface markers listed in Table 1 of U.S. Pat. No. 6,004,528 or Tables1, 2, or 3 of U.S. patent application Ser. No. 09/468,286, and U.S.Patent Application Publication Nos. 2006/0083682, 2007/0036800,2007/0036801, 2007/0036802, 2007/0041984, 2007/0036803, and2007/0036804, each of which is incorporated by reference herein. Inaccordance with this example, the pharmaceutical pack or kit comprisesone or more antibodies which bind to cancer stem cell surface markers,wherein each antibody binds to a different epitope of the cancer stemcell surface marker and/or binds to the cancer stem cell surface markerwith a different affinity.

For antibody based kits, the kit can comprise, for example: (1) a firstantibody (which may or may not be attached to a solid support) whichbinds to a cancer stem cell surface marker protein; and, optionally, (2)a second, different antibody which binds to either the cancer stem cellsurface marker protein bound by the first antibody, or the firstantibody and is conjugated to a detectable label (e.g., a fluorescentlabel, radioactive isotope or enzyme). The antibody-based kits may alsocomprise beads for conducting an immunoprecipitation. Each component ofthe antibody-based kits is generally in its own suitable container.Thus, these kits generally comprise distinct containers suitable foreach antibody. Further, the antibody-based kits may compriseinstructions for performing the assay and methods for interpreting andanalysing the data resulting from the performance of the assay. As anexample, a kit may include an anti-CD34 antibody for positive selection,an anti-CD38 antibody for negative selection, and an anti-CD123 antibodyfor positive selection to isolate and/or quantify and/or assist in thedetermination of the amount of leukemia cancer stem cells (which areCD34+/CD38−/CD123+).

For nucleic acid micoarray kits, the kits generally comprise (but arenot limited to) probes specific for certain genes attached to a solidsupport surface. In other examples, the probes are soluble. In one suchexample, probes can be either oligonucleotides or longer length probesincluding probes ranging from 150 nucleotides in length to 800nucleotides in length. The probes may be labeled with a detectablelabel. The microarray kits may comprise instructions for performing theassay and methods for interpreting and analysing the data resulting fromthe performance of the assay. The kits may also comprise hybridizationreagents and/or reagents necessary for detecting a signal produced whena probe hybridizes to a cancer stem cell surface marker nucleic acidsequence. Generally, the materials and reagents for the microarray kitsare in one or more containers. Each component of the kit is generally inits own suitable container.

For Quantitative PCR, the kits generally comprise pre-selected primersspecific for certain cancer stem cell surface marker nucleic acidsequences. The Quantitative PCR kits may also comprise enzymes suitablefor amplifying nucleic acids (e.g., polymerases such as Taq), anddeoxyribonucleotides and buffers needed for the reaction mixture foramplification. The Quantitative PCR kits may also comprise probesspecific for the nucleic acid sequences associated with or indicative ofa condition. The probes may or may not be labeled with a flourophore.The probes may or may not be labeled with a quencher molecule. In someexamples, the Quantitative PCR kits also comprise components suitablefor reverse-transcribing RNA including enzymes (e.g., reversetranscriptases such as AMV, MMLV and the like) and primers for reversetranscription along with deoxynucleotides and buffers needed for thereverse transcription reaction. Each component of the quantitative PCRkit is generally in its own suitable container. Thus, these kitsgenerally comprise distinct containers suitable for each individualreagent, enzyme, primer and probe. Further, the quantitative PCR kitsmay comprise instructions for performing the assay and methods forinterpreting and analysing the data resulting from the performance ofthe assay.

A kit can optionally further comprise a predetermined amount of anisolated cancer stem cell surface marker polypeptide or a nucleic acidencoding a cancer stem cell surface marker, e.g., for use as a standardor control. The diagnostic methods of the present invention can assistin conducting or monitoring a clinical study. In accordance with thepresent invention, suitable test samples, e.g., of serum or tissue,obtained from a subject can be used for diagnosis.

Based on the results obtained by use of the pharmaceutical pack or kit(i.e. whether the cancer stem cell amount has stabilised or decreased),the medical practitioner administering the cancer therapy or regimen maychoose to continue the therapy or regimen. Alternatively, based on theresult that the cancer stem cell amount has increased, the medicalpractitioner may choose to continue, alter or halt the therapy orregimen.

Any reference to prior art documents in this specification is not to beconsidered an admission that such prior art is widely known or formspart of the common general knowledge in the field.

As used in this specification, the words “comprises”, “comprising”, andsimilar words, are not to be interpreted in an exclusive or exhaustivesense. In other words, they are intended to mean “including, but notlimited to”.

The invention is further described with reference to the followingexamples. It will be appreciated that the invention as claimed is notintended to be limited in any way by these examples.

EXAMPLES Example 1: Materials and Methods Materials Antibodies

The antibodies used for immunohistochemistry, their optimised dilutionsand sources are listed in alphabetical order in Table 1.

TABLE 1 Antibodies, Dilution and Sources Optimised Dilution - OptimisedOptimised DAB Dilution - Antibody Autostainer Fluorescent Dilution -Control Antibody Species Source IHC IHC WB tissue Santa Cruz RabbitNewcastle 1:300 1:200 Seminoma Biotechnology upon Tyne, Nanog UK AbcamRabbit Billerica, 1:1000 1:1000 Placenta Renin Ma, USA Receptor CellMarque Mouse Santa Cruz, 1:300 Seminoma Oct-4 Monoclonal Ca, USA ThermoFisher Rabbit Santa Cruz, 1:2000 1:500 Colon Scientific polyclonalCalifornia, Cancer Sox2 USA Life Mouse Rockford, 1:5000 UbiquitouslyTechnologies monoclonal Il, USA expressed β-actin Abcam Goat anti-Carlsbad, 1:500 Secondary Rabbit Ca, USA antibody Abcam Sheep anti-Carlsbad, 1:500 Secondary Mouse Ca, USA antibody Abcam Goat anti-Carlsbad, 1:2000 Secondary Mouse Ca, USA HRP conjugated antibody AbcamSheep anti- Carlsbad, Secondary goat Ca, USA HRP conjugated antibodyAbcam Goat anti- Carlsbad, 1:2000 Secondary Rabbit Ca, USA HRPconjugated antibody Life Goat anti- Cambridge, 1:500 Technologies MouseEngland Alexafluor Fluorophore 488 Life Donkey Cambridge, 1:500Technologies, anti-sheep England Alexafluor Fluorophore 488 Life GoatAnti- Carlsbad, 1:2000 Technologies Mouse Ca, USA Alexafluor 555 LifeGoat anti- Carlsbad, 1:500 Technologies Rabbit Ca, USA Alexafluor 594Life Chicken Carlsbad, 1:500 Technologies anti-Goat Ca, USA Fluorophore647 Life Goat Anti- Carlsbad, 1:2000 Technologies, Rabbit Ca, USAAlexafluor 647 Abcam Goat Anti- Carlsbad, 1:2000 Fluorophore Mouse Ca,USA Abcam Goat Anti- Carlsbad, 1:2000 Fluorophore Rabbit Ca, USA Bio-Rad— Cambridge, — — Prestained England SDS-PAGE Standards, Broad RangeBio-Rad — Cambridge, — — Precision Plus England Protein ™ Dual XtraStandards

Chemicals and Reagents

The Chemicals and Reagents used in this study and their sources arelisted in Table 2.

TABLE 2 Chemicals and Reagents Emsure Sodium Chloride Darmstadt, GermanyEmsure Potassium Chloride Darmstadt, Germany Sigma-Aldrich SodiumCitrate, pH 6 St Louis, Mo, USA 2.491 g Citrate, 1000 mL ddH2O, pH 6Sigma-Aldrich Tween 20 ™ St Louis, Mo, USA Sigma-Aldrich SodiumBorohydride St Louis, Mo, USA Sigma-Aldrich Bovine Serum Albumin (BSA)St Louis, Mo, USA Merck 1,4 Dithiothrietol (DTT) Darmstadt, Germany GEHealthcare 2-D Quant Kit Piscataway, NJ, USA BSA Standard CopperSolution Colour Reagent A Colour Reagent B Precipitant Co-PrecipitantLeica Micromount, Invitrogen Gold Antifade Nussloch, Germany containingDAPI Leica Surgipath^(R) Micromount^(R) Nussloch, Germany MountingMedium

Buffers, Solutions and Stains

The buffers, solutions and stains used in this study are listed in Table3.

TABLE 3 Buffers, solutions and stains Sigma Aldrich SAFC^(R) TrisHydrochloride St Louis, Mo, USA Sigma Aldrich Tween20 ™ St Louis, Mo,USA Tris-Buffered Saline (TBS), pH 7.6 10X: 80 g (Sigma Aldrich) NaCl, 2g (Sigma Aldrich) KCl, 30 g Tris Base, 1000 mL ddH2O TBS/Tween (TBST,TBS + 1% Tween20) Phosphate-Buffered Saline (PBS), pH 7.4 10X: 80 g(Sigma Aldrich) NaCl, 2 g (Sigma Aldrich) KCl, 14.4 g Na₂HPO₄. 2.4 gKH₂PO₄, 1000 mL ddH₂0 PBS/Tween (PBST, PBS + 1% Tween20) Bio-RadReadyPrep ™ Bio Protein Extraction kit Hercules, Ca, USA (Reducing): 5.6mL ReadyPrep Proteomic grade water One vial containing 5.6 g BufferPowder 10 uL tributyl phosphine (TBP, reducing agent) Transfer contentsof 1 mL ampoule to screw cap storage vial provided Store remaining at−80 C. Sigma Aldrich Life Science RIPA buffer St Louis, Mo, USA ThermoFisher scientific Halt ™ Protease & Rockford, Il, USA PhosphataseInhibitor Cocktail Bio-Rad 2x Laemmli Sample Buffer Hercules, Ca, USABio-Rad 2-mercaptoethanol Hercules, Ca, USA Bio-Rad 10X Tris/Glycine/SDSBuffer Hercules, Ca, USA SDS-PAGE Running Buffer Life Technologies ™,Molecular Probes^(R) Qubit^(R) Carlsbad, Ca, USA Protein assay Kit:Qubit^(R) Protein Buffer Qubit^(R) Protein reagent Life Technologies,Novex, iBind ™ Solution Carlsbad, California, Kit USA iBind ™ 5x BufferiBind ™ 100x Additive Life Technologies, Novex, iBind ™ FD SolutionCarlsbad, California, Kit USA iBind ™ 5x Buffer iBind ™ 100x AdditiveiBind ™ 10% SDS Life Technologies ™ Qubit* Protein Standards Eugene, Or,USA Bio-Rad Clarity ™ Western ECL Substrate Kit: Hercules, Ca, USALuminol/enhancer solution Peroxide solution Leica Bond ™ Dewax Solution.Nussloch, Germany

The kits used for the autostainer immunohistochemistry and their sourcesare listed in Table 4.

TABLE 4 Immunohistochemistry Autostainer Kits Autostainer Kit Cat#Source Cat#AR9222 Nussloch, Germany Leica Bond ™ Primary AntibodyCat#AR9352 Nussloch, Germany Diluent Leica Bond ™ Enzyme PretreatmentCat#AR9551 Nussloch, Germany Kit Leica Bond ™ Epitope RetrievalCat#AR9961 Nussloch, Germany Solution 1 Leica Bond ™ Epitope RetrievalCat#AR9640 Nussloch, Germany Solution 2 Leica Bond ™ Intense R DetectionCat#DS9263 Nussloch, Germany Leica Polymer Bond ™ Intense Cat#DS9390Nussloch, Germany Refine Red Detection Leica Polymer Bond ™ IntenseCat#DS9800 Nussloch, Germany Refine Detection

Methods Formalin-Fixed Paraffin-Embedded Section Preparation

4 um sections of formalin fixed and paraffin-embedded SCCOT sectionswere cut using a microtome, stretched in a water-bath at 40° C.,transferred onto glass slides and dried at room temperature overnight.

H&E Staining

H&E staining was performed on one formalin fixed and paraffin embeddedslide per patient.

H&E Staining Analysis

H&E stained slides were used for initial grading of lesions intowell-differentiated, moderately-differentiated and poorly-differentiatedgroups by a consultant pathologist. They were subsequently used as areference for tissue morphology and orientation of IHC staining patternswithin the specimens.

Automated Fluorescent Immunohistochemistry Procedure

Automated fluorescent immunostaining with either double stains, triplestains, or quadruple stains was performed according to protocolsprovided by the manufacturer, using a Leica Bond RX ImmunohistochemistryAutostainer, Nussloch, Germany.

Automated DAB Immunohistochemistry Staining Procedure

Automated DAB immunostaining with single stains was performed accordingto protocols provided by the manufacturer, using a Leica Bond RXImmunohistochemistry Autostainer, Nussloch, Germany.

Visualisation, Photography, and Image Analysis

DAB stained slides were viewed with an Olympus BX53 microscope, and anOlympus SC100 microscope camera.

DAB immunostained slides were viewed and captured using an Olympus BX53Microscope, and Olympus SC100 Microscope Camera. Qualitative analyses ofthe images were described as either non-staining, weak-positivestaining, strong positive staining. Observations about the location ofpositive staining within the tissue, morphology of positive cells, andapparent location of staining within positive staining cells were alsonoted.

Fluorescent immunostained slides were viewed and captured using anOlympus FV1200 biological confocal laser-scanning microscope from Tokyo,Japan.

Qualitative analyses of the images were described as eithernon-staining, staining or co-staining.

Localisation of marker expression within cells was analysed using FV-10software, generating the relative intensity of expression of multiplemarkers across the diameter of single cells. Expression was separatedinto nuclear, cytoplasmic, and membranous regions. The average stainingintensity within each of these portions was calculated in 9 individualcells per patient. This allowed generalisations to be made regarding theprecise localisation of staining within the cells of that patient. Forexample 70% nuclear, 30% cytoplasmic, 0% membranous.

Western Blotting Sample Handling and Preparation

Surgical specimens were transferred directly from the operating theatreto the GMRI laboratory where they were placed into Cryovials and snapfrozen with liquid nitrogen and stored at −80° C.

Snap-frozen tissues were taken from the freezer and dissected to anappropriate size (40-80 mg). The mass of the sample to be used wasrecorded, and any remaining tissue was immediately returned to theoriginal Cryovial and stored at −80° C. for future research. Sampleswere kept on ice as much as possible.

Samples were added to individual Eppendorf tubes containing RIPA bufferand a protease inhibitor, 10 mg/500 uL/5 uL, and then transferred to aglass mortar and ground on ice with a pestle, until homogenized. Thetissue suspension was then returned to the eppendorf tube, incubated onice for 5 minutes, then centrifuged for at 13300 rpm 4° C. for 20minutes. The supernatant was then transferred to a new Eppendorf tube,and stored on ice. The pellet was discarded.

Protein Assay

Qubit™ Working Solution was prepared by diluting 1 ul Qubit™ Reagentwith 199 ul Qubit™ Buffer to make 200 uL for each of the standards andsamples. Three protein standards were then prepared using 10 ul (proteinstandards) added to 190-199 ul Qubit™ Working Solution. Standards werethen vortexed for 2-3 seconds, and incubated at room temperature for 2minutes. Each of the standards was then placed into the Qubit™ 2.0fluorometer sequentially to derive a standard curve.

Experimental protein samples were prepared using 10 uL of lysate addedto 190 uL of Qubit™ Working Solution to make up 200 ul. Samples werethen read using the Qubit™ 2.0 Fluorometer, and the proteinconcentration in the original stock sample was calculated using thedilution calculator feature.

If the sample protein concentration could not be read n because therewas excess protein in the sample, the lysate was diluted and the processrepeated until a satisfactory reading was obtained.

Tissue lysate samples were then aliquoted into eppendorf tubes to reducecontamination risk and damage caused by repeated freeze/thaw cycles andthen stored at −80° C. for later use.

Lysate samples were retrieved from the freezer, and kept on ice. Thevolume of each sample was calculated based on the protein concentrations(to allow loading of equal amounts of protein per well on the gel) andthen Extraction buffer was added to these so that each sample was ofequal volume (10 uL).

Working solution (9.5 uL of Laemmli buffer and 0.5 uL β-mercaptoethanol)was prepared to make 10 uL per sample. 10 uL sample and 10 uL workingsolution was then boiled on a heating block for 10 minutes.

Gel Electrophoresis

Laemmli SDS-PAGE running buffer was prepared by adding 100 mL 10× stockto 900 mL ddH20.

The comb and tape were removed from a mini-SDS PAGE gel and theMini-Protean tetra cell was assembled and the inner and outer chamberswere filled with 1× running buffer.

2.5 uL of the protein standard (molecular weight markers) was loadedinto gel well 1 and 20 uL of the protein samples were loaded into theremaining wells.

The gel was then run at 110V for 5 minutes until the samples entered thegel and the voltage was then increased to 200V to for 30 minutes. Thegel was then removed, and the wells and bottom of the gel (containingloading dye) were cut off with a scalpel.

Semi-Dry Transfer of Gel to Membrane

The transfer sandwich was assembled in the Bio-Rad Trans-Blot Turbo™Transfer system, in the correct order: filter paper, PVDF membrane, gel,filter paper. A small amount of 1× running buffer was applied to wet themembrane and the roller was used to remove any trapped air bubbles. Thechamber was then closed and the transfer initiated with the correctsettings (TURBO, 1 MINI GEL (1.3 A, 25V, 7 min) A: Run) and run for 7minutes. The blot was then removed from the sandwich and placed brieflyin working solution.

Immunodetection

1× iBind™ solution was prepared with 23.7 mL ddH20, and 300 uL Additiveand 6 mL iBind™ buffer. The blotted membrane was immersed for 10 minutesin 10 mL of the iBind™ solution on a rocking platform. An iBind™ Cardwas placed on the stage of the iBind™ Western Device, and 5 mL of 1×iBind™ solution was pipetted evenly across the flow region, with anadditional 1 mL of iBind™ solution pooled in the centre of the card. Theblotted membrane was then placed on the iBind™ Card with the proteinside down and the low molecular weight region closest to the stack. Theblotting roller was then used to remove any air bubbles, and the lid wasclosed. Antibodies and 1× iBind™ solution were then added to the wellsin the following sequence. 1: Primary antibody (5× concentration) in 2mL of working solution, 2: 2 mL iBind™ solution, 3: HRP secondaryantibody (5× concentration) in 2 mL of working solution, 4: 6 mL iBind™solution, and incubated for 2.5 hours or overnight at room temperature.

Imaging and Data Analysis

The blotted membrane was rinsed in tap water for 5 minutes on a rockingplatform. The chemiluminescent substrate kit (Clarity™ Western ECLSubstrate Kit) was prepared in a 1:1 ratio: 7 mL Luminol enhancersolution and 7 mL peroxide solution and incubated for 15 minutes on arocking platform. The chemiluminescent signals were then captured usinga Bio-Rad ChemiDoc™ MP Imaging System, and Bio-Rad Image Lab™ SoftwareVersion 5.0.

Re-Probing

Blotted membranes were stored in TBST at 4 degrees C. When required,blotted membranes were re-probed for an additional marker. The blottedmembrane was washed in tap water for 10 minutes on a rocking platform,and then incubated in 10 mL of iBind™ solution for 10 minutes on arocking platform. iBind™ Card, iBind™ working solution and antibodieswere prepared as previously described, with the exception of usingfluorescent secondary antibodies rather than HRP secondary antibodies.The membrane was then incubated for 3.5 hours.

Protein Quantification

Protein extracted from an initial sample of poorly differentiated tongueSCC sample. Protein quantification was performed using the 2-D Quantkit. Protein standards were prepared by placing 0 uL, 10 uL, 20 uL, 30uL, 40 uL, 50 uL of BSA solution into Eppendorf tubes. OTSCC and controltissue (placenta) samples were prepared covering a range of proteinlysates (2 uL, 5 uL, 15 uL, 30 uL) in Eppendorf tubes. 500 uL ofprecipitant solution was added to each, and each tube was vortexed for 5seconds. 500 uL of co-precipitant was then added to each tube and againvortexed for 5 seconds. All protein standards and solutions were thencentrifuged at 13,300 rpm for 15 minutes. The supernatant was poured offand discarded and the tubes were again centrifuged briefly to move allremaining solution from the bottom of the tubes. This solution was thenpipetted off, taking care not to disturb the pellet.

500 uL copper solution was prepared for each of the protein standardsand samples, 1:5 copper solution:ddH2O. 500 uL copper solution was thenadded to each of the tubes, and vortexed for 5 seconds, to re-dissolvethe precipitated protein.

1000 uL of colour reagent solution was prepared for each of the proteinstandards and samples, 1:200 colour reagent A:colour reagent B. 1000 uLof colour reagent solution was added to each tube and left to developfor 5 minutes. 200 uL each of ddH20, standard solutions and samples wereloaded sequentially to a well plate.

The well plate was placed in a plate-reader. A standard curve wasgenerated from the standard solutions, and the absorbance of sampleswere read at 480 nm.

The standard curve was then generated and the samples read against it,to give a total protein concentration in mg/mL in the stock sampleprotein lysate.

Example 2: Expression Analysis in Squamous Cell Carcinoma of Oral Cavity

The following markers were analysed in tissue samples obtained frompatients having squamous cell carcinoma of oral tongue (SCCOT).

1. Embryonic Stem Cell Markers

OCT4:

Embryonic stem cell marker, associated with maintenance of pluripotencyand self-renewal, not expressed in normal adult tissues. Transcriptionfactor with expression normally confined to the nucleus although somecytoplasmic expression in cancer cells has been noted in the literature(also noted in our results).

NANOG:

Embryonic stem cell marker, associated with maintenance of pluripotencyand self-renewal, expression controlled by SOX2 and OCT4. Transcriptionfactor with expression normally confined to nucleus, but again somecytoplasmic expression in cancer cells has been noted in the literature(also noted in our results).

SOX2:

Embryonic stem cell marker, associated with maintenance of pluripotencyand self-renewal. Transcription factor with expression mainly in thenucleus but again some cytoplasmic expression in cancer cells has beennoted in the literature (also noted in our results).

pSTAT3:

Activated Signal Transducer and Activator of Transcription. Known stemcell marker associated with maintenance of pluripotency, self-renewal.Constitutive activation of pSTAT3 is recognised in cancer, playing a keyrole in control of cell-cycle progression and anti-apoptosis andangiogenesis.

The results of the marker expression and marker co-localisation analysesperformed on squamous cell carcinoma of oral tongue (SCCOT) samples aresummarised in Table 5, below.

TABLE 5 Summary of marker expression within moderately differentiatedsections of SCC of the oral tongue (to date). Investigation ofco-localisations. Antibody Antibody against against protein protein Co-Autostainer marker 1 marker 2 expression? Interpretation AutostainerCD44 NANOG Yes Primitive cancer (Ms)* (Rb)* stem cell populationAutostainer CD44 SOX2 Yes Primitive cancer (Ms)* (Rb)* stem cellpopulation Autostainer EMA SOX2 No EMA cells are more (Ms)* (Rb)*differentiated (downstream) of SOX2 positive cells *(Rb) primaryantibody raised in rabbit (Ms) primary antibody raised in mouse

8. Positive Controls

Positive control tissues (target protein specific) were identified,tested and then stained simultaneously with experimental tissues on theautostainer.

9, Results

The results of these data show:

-   -   1. Definitive co-localisation of cancer stem cell markers within        tongue cancer. The proteins that have been co-localised within        this cancer stem cell population to date are: NANOG, OCT4, SOX2,        CD44, LYVE-1, VEGFR-3 and ACE.    -   2. Cancer stem cells do not co-express the epithelial cancer        markers EMA, p63 or cytokeratins 5,6,8,18. This suggests that        these cells are more primitive (upstream) than the bulk cancer        cells that do express these markers.    -   3. For the first time, lymphatic markers (LYVE-1 and VEGFR-3)        and cancer stem cell markers (CD44) and embryonic stem cell        markers (NANOG, OCT4, SOX2) have been shown to be co-expressed.        This has interesting implications for understanding the biology        of this cell population, and suggests that these cells may be        actively involved in driving metastasis via lymphatic channels        as distinct from the blood vessels, and also supports our        hypothesis that these cancer stem cells are truly pluripotent        and capable of de novo lymphangiogenesis (in addition to        self-renewal and formation of the bulk tumour cells).    -   4. Expression of ACE by the cancer stem cell population is also        novel, and supports the hypothesis that the renin-angiotensin        system (RAS) is involved in regulation of cancer stem cells in        some capacity.

Example 3: Expression Analysis Other Cancers

To perform co-expression studies for the expression of the embryonicstem cell marker, OCT4, and Renin Receptor, paraffin sections werestained sequentially for the presence of the two proteins. The imageswere captured using the Olympus FV-1200 Confocal Microscope and analysedusing the Olympus Fluoview FV1000 software. A representative cancer stemcell from each of the cancer systems examined were selected and across-sectional line drawn through the middle of it, with the relativeexpression levels of OCT4, the Renin Receptor relative to the cellnucleus, shown by the expression of 4′,6-diamidino-2-phenylindole(DAPI). This evident from the data in the accompanying figures by cancertype, namely: OTSCC (FIGS. 4A, 4B), melanoma (FIGS. 7A, 7B), sarcoma(FIGS. 9A, 9B), bowel cancer (FIGS. 11A, 11B), brain cancer (FIGS. 13A,13B), breast cancer (FIGS. 15A, 15B), lung cancer (FIGS. 17A, 17B), Bcell lymphoma (FIGS. 19A, 19B), and kidney (FIGS. 21A, 21B), thyroidcancer (23A, 23B), chronic lymphocytic cancer (25A, 25B), skin squamouscell carcinoma (27A, 27B), prostate cancer (29A, 29B).

The Pro/Renin receptor is a cell surface and cytoplasmic cellularprotein detected at about 39 KDa, this protein is then able to becleaved intracellularly by a number of proteins, such as but not limitedto Furin and Cathepsin B (e.g. Wang et al. (1991) J. Biol. Chem.266(19): 12633-12638). The cleaved product is a truncated form of theoriginal protein, which is about 25 KDa in size. Both the full lengthand the cleaved Pro/Renin forms are shown in FIG. 5. The latter cleavedform is actively secreted into the extracellular fluid, eventuallymaking its way into bodily fluids such as blood, lymph. Accordingly,this marker may be used as a biomarker of cancer, and in particularcancer with an associated cancer stem cell population(s).

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made withoutdeparting from the scope of the invention as defined in the claims.Furthermore, where known equivalents exist to specific features, suchequivalents are incorporated as if specifically referred in thisspecification.

1. A method for preventing, treating, or managing cancer in a patient inneed thereof, the method comprising (a) detecting expression of OCT4,SOX2, NANOG, c-Myc, and KLF4 on cancer stem cells from the patient, and(b) administering one or more therapeutic agent(s) to the patient in anamount sufficient to selectively eradicate, or inhibit the growth,proliferation and/or differentiation of cancer stem cells in a tumourwithin the cancer, wherein the therapeutic agent is selected from thegroup consisting of Direct Renin Inhibitors (DRIs),Angiotensin-Converting Enzyme Inhibitors (ACEIs), Angiotensin ReceptorBlockers (ARBs), Beta-Blockers, Cyclo-oxygenase 2 Inhibitors, ChymaseInhibitors, Inhibitors of Cathepsin B, Cathepsin D and Cathepsin G,Calcium, Vitamin D, and Calcium Channel Blockers. 2.-5. (canceled)
 6. Amethod according to claim 1, wherein the cancer is selected from thegroup consisting of squamous cell carcinoma of the head and neck(including the upper aerodigestive tract), squamous cell carcinoma ofthe skin, melanoma, lung cancer, breast cancer, kidney cancer, braincancer, bowel cancer, thyroid cancer, prostate cancer, lymphoma,leukemia and sarcomas.
 7. A method according to claim 6, wherein thecancer is squamous cell carcinoma of the head and neck.
 8. A methodaccording to claim 6, wherein the squamous cell carcinoma of the headand neck is located in the aerodigestive track.
 9. A method forpreventing, treating, or managing cancer in a patient in need thereof,the method comprising (a) detecting expression of SOX2, NANOG, c-Myc,and KLF4 on cancer stem cells from the patient, and (b) administeringone or more therapeutic agents to the patient in an amount sufficient toselectively eradicate or inhibit the growth, proliferation and/ordifferentiation of cancer stem cells in a tumour associated with thecancer, wherein the therapeutic agent is selected from the groupconsisting of Direct Renin Inhibitors (DRIs), Angiotensin-ConvertingEnzyme Inhibitors (ACEIs), Angiotensin Receptor Blockers (ARBs),Beta-Blockers, Cyclo-oxygenase 2 Inhibitors, Chymase Inhibitors,Inhibitors of Cathepsin B, Cathepsin D and Cathepsin G, Calcium, VitaminD, and Calcium Channel Blockers. 10.-15. (canceled)
 16. The method ofclaim 9, wherein step (a) comprises detecting lack of expression ofOCT4.
 17. The method according claim 9, wherein the cancer is selectedfrom the group consisting of squamous cell carcinoma of the head andneck, squamous cell carcinoma of the skin, melanoma, lung cancer, breastcancer, kidney cancer, brain cancer, bowel cancer, thyroid cancer,prostate cancer, lymphoma, leukemia and sarcomas.
 18. The methodaccording to claim 9, wherein the cancer is squamous cell carcinoma ofthe head and neck.
 19. The method according to claim 9, wherein thesquamous cell carcinoma of the head and neck is located in theaerodigestive tract.